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J Bacteriol. 2010 June; 192(11): 2722–2728.
Published online 2010 March 19. doi:  10.1128/JB.00112-10
PMCID: PMC2876504

Characterization of the Pseudomonas aeruginosa Transcriptional Response to Phenylalanine and Tyrosine[down-pointing small open triangle]

Abstract

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen often associated with chronic infections in the lungs of individuals with the heritable disease cystic fibrosis (CF). Previous work from our laboratory demonstrated that aromatic amino acids within CF lung secretions (sputum) not only serve as carbon and energy sources but also enhance synthesis of the cell signaling molecule Pseudomonas quinolone signal (PQS). The present study investigates the role of the aromatic amino acid-responsive regulator PhhR in mediating these phenotypes. Transcriptome analysis revealed that PhhR controls four putative transcriptional units (phhA, hpd, hmgA, and dhcA) involved in aromatic amino acid catabolism; however, genes involved in PQS biosynthesis were unaffected. The phhA, hpd, hmgA, and dhcA promoters were mapped by primer extension, and purified His6-PhhR was shown to bind the phhA, hpd, and dhcA promoters in vitro by use of electrophoretic mobility shift assays. Our work characterizes a transcriptional regulator of catabolic genes induced during P. aeruginosa growth in CF sputum.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen commonly found in soil and water. It is notorious for causing persistent, chronic lung infections in individuals with the genetic disease cystic fibrosis (CF). A critical symptom of CF is a buildup of thick mucus (sputum) in the lungs, which inhibits the ability to clear invading pathogens (6). Additionally, sputum represents an excellent growth substrate for several bacteria, including P. aeruginosa (10). Due to natural resistance to most conventional antimicrobials, P. aeruginosa infections are particularly difficult to treat and are the leading cause of morbidity and mortality in individuals with CF (7).

To better understand the physiology of P. aeruginosa during growth in the CF lung, we previously developed a defined synthetic CF sputum medium (SCFM) that mimics the nutritional environment of CF sputum (19). Consistent with growth in authentic CF sputum, P. aeruginosa produces higher levels of the cell signaling molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]) in SCFM (19, 21). PQS is a quorum sensing signal (23) essential for production of a range of secreted virulence factors, including phenazines and hydrogen cyanide (9). Interestingly, removal of aromatic amino acids from SCFM decreased PQS production approximately 5-fold, implicating these amino acids as key mediators of enhanced PQS production in CF sputum (19). Further study revealed that the aromatic amino acids phenylalanine and tyrosine, but not tryptophan, were the primary inducers of PQS biosynthesis in SCFM (19, 21). While tryptophan was recently reported to enhance production of PQS (8), this amino acid is likely not important in CF sputum, as it is present at extremely low levels (~10 μM) (19).

In addition to enhancing PQS production, phenylalanine and tyrosine also serve as important carbon sources for P. aeruginosa in CF sputum (19). 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).

The present study investigates the role of PhhR in mediating two important P. aeruginosa phenotypes observed during growth in CF sputum: catabolism of phenylalanine/tyrosine and phenylalanine/tyrosine-mediated induction of PQS. Transcriptome analysis revealed that PhhR controls four putative transcriptional units involved in aromatic amino acid catabolism; however, genes involved in PQS biosynthesis were unaffected. Promoters for several identified genes were mapped, putative binding sites for PhhR were identified using in silico analysis, and the ability to specifically bind these promoter regions was determined using electrophoretic mobility shift assays (EMSAs).

MATERIALS AND METHODS

Bacterial strains and media.

P. aeruginosa strain PA14 and the isogenic phhR mutant were obtained from the PA14 nonredundant transposon mutant library (15; http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi). The transposon insertion site in phhR was confirmed by PCR. P. aeruginosa was routinely cultured on tryptic soy agar (14). Escherichia coli DH5α was used as the recipient for transformation and was cultured on LB Miller broth/agar (Fisher Scientific). Cultures were grown at 37°C with shaking at 250 rpm. Antibiotics were used at the following concentrations, unless otherwise noted: ampicillin, 50 μg/ml for E. coli; and carbenicillin, 300 μg/ml for plasmid selection and 25 μg/ml for plasmid maintenance in P. aeruginosa.

P. aeruginosa was also grown in defined SCFM (19). SCFM normally contains 0.8 mM tyrosine, 0.5 mM phenylalanine, and 10 μM tryptophan; however, when SCFM without aromatic amino acids was used, equimolar serine was added in place of these amino acids. Serine was chosen because it has been shown previously to not affect PQS production (19, 21). To evaluate growth of P. aeruginosa with tyrosine or phenylalanine as a carbon and energy source, a MOPS (morpholinepropanesulfonic acid)-buffered salts base (50 mM MOPS, pH 7.2, 93 mM NH4Cl, 43 mM NaCl, 3.7 mM KH2PO4, 1 mM MgSO4, 3.5 μM FeSO4·7H2O, 2 mM proline) was supplemented with 10 mM tyrosine or 10 mM phenylalanine. Proline was added to reduce the considerable lag in growth commonly observed when phenylalanine/tyrosine is used as a sole carbon and energy source.

DNA manipulations.

Standard methods were used to manipulate plasmids and DNA fragments (2). Restriction endonucleases and DNA modification enzymes were purchased from New England Biolabs. Chromosomal DNA from P. aeruginosa was isolated using DNeasy Tissue kits (Qiagen), and plasmid isolations were performed using QIAprep spin miniprep kits (Qiagen). DNA fragments were purified using QIAquick Mini-Elute PCR purification kits (Qiagen), and PCR was performed using the Expand Long Template PCR system (Roche).

PQS extraction.

Overnight cultures of P. aeruginosa PA14 or the phhR mutant grown in SCFM were subcultured into fresh SCFM to an optical density at 600 nm (OD600) of 0.05. Cells were grown to exponential phase (OD600 of 0.45 to 0.55), pelleted by centrifugation at 9,000 × g for 5 min, washed twice with carbon-free SCFM, and starved for 2 h in carbon-free SCFM. Starved cultures were used to inoculate SCFM or SCFM without aromatic amino acids to an OD600 of 0.01. Cultures were allowed to reach near-maximum growth yields (OD600 of ~3) and were extracted twice with an equal volume of acidified ethyl acetate (150 μl acetic acid per liter ethyl acetate; Fisher). Extracts were dried under a constant stream of N2 gas, resuspended in methanol (Fisher), and analyzed by thin-layer chromatography (TLC) as described previously (18, 19, 23). PQS was identified by comigration with 500 ng synthetic PQS standard. PQS on TLC plates was visualized by fluorescence after excitation with long-wave UV light, using a G:Box gel imager (Syngene).

Global expression profiling.

For Affymetrix GeneChip analysis, P. aeruginosa PA14 was grown in SCFM or SCFM without aromatic amino acids, and the phhR mutant was grown in SCFM. To prepare cells for RNA extraction, exponentially growing cells were diluted to an OD600 of 0.001 and allowed to grow to an OD600 of 0.35 to 0.45. Cultures were mixed 1:1 with RNALater (Ambion), and total RNA was isolated. DNA contamination within RNA samples was removed by DNase treatment (Promega) and monitored by PCR amplification of the rplU gene as previously described (17, 19-21, 25). RNA integrity was monitored by agarose gel electrophoresis. cDNA synthesis from total RNA and cDNA fragmentation and labeling were performed as previously described (17, 19-21, 25). Processing of Affymetrix P. aeruginosa GeneChips was performed at the University of Iowa DNA Facility. All experiments were performed in duplicate, and data were analyzed using GeneChip operating software, version 1.4. Differentially regulated genes were identified by pairwise comparisons (4 or 6 total) of all GeneChips (P ≤ 0.05).

Complementation of the P. aeruginosa phhR mutant.

The phhR gene was PCR amplified from P. aeruginosa PA14 chromosomal DNA by using the primers phhR-comp-for (5′-TCCCCCGGGGAACGACAACACNNNNNCACGCC-3′) and phhR-comp-rev (5′-TCCCCCGGGCCGCCGTTTCTTTCCCAGCCTG-3′). The phhR-comp-for primer was designed such that the native Shine-Dalgarno sequence of phhR was replaced with N5. The resulting 1,628-bp fragments were cloned into the pGEM-T Easy vector (Promega) per the manufacturer's instructions. Plasmids were isolated from 10 pooled white colonies and digested with SmaI to excise phhR. The phhR DNA fragments were gel purified and then ligated into SmaI-digested pUCP18 (29). Resulting pUCP18 plasmids containing phhR were purified and transformed into the P. aeruginosa phhR mutant by MgCl2 transformation (30). Transformants were screened for the ability to grow with tyrosine as a carbon source. One plasmid that restored growth with tyrosine was identified (pKP-phhR). The sequence of the phhR gene in pKP-phhR was confirmed by DNA sequencing at the University of Texas Institute for Cell and Molecular Biology DNA Facility. In pKP-phhR, phhR expression is controlled by a constitutive lac promoter.

Primer extension analysis.

To obtain RNA for primer extension analyses, P. aeruginosa was grown to late exponential phase in SCFM (OD600 of 1.3) and mixed 1:1 with RNALater (Ambion). Total RNA was isolated using an RNeasy Mini kit (Qiagen), and DNA contamination and RNA integrity were monitored as described above for Affymetrix GeneChip analysis. Primer extension was performed using fluorescently 6-carboxyfluorescein (FAM)-labeled primers as previously described (4, 16). Briefly, 1 μl of a 0.4 μM 5′-FAM-labeled primer was added to 20 to 30 μg total RNA in a final volume of 20 μl and incubated at 70°C for 10 min. Reaction mixtures were quickly chilled in an ice-water bath and then incubated at 65°C for 20 min. The temperature was shifted to 42°C, and reagents for cDNA synthesis (SuperScript II system [Invitrogen]; 8 μl 5× buffer, 4 μl 0.1 M dithiothreitol [DTT], 4 μl 10 mM deoxyribonucleotides, 4 μl SuperScript II enzyme) were added. Reaction mixtures were incubated at 42°C for 2 h, ethanol precipitated, and submitted for DNA sizing analysis at the University of Oklahoma Health Sciences Center Laboratory for Genomics and Bioinformatics. Some reaction mixtures were treated with 1 μl RNase H (Invitrogen) at 37°C for 20 min prior to precipitation. Primers used for primer extension are shown in Fig. Fig.2.2. When more than one fluorescent peak was present, the highest peak, which corresponds to the major primer extension product, was reported. Primer extension analysis of each gene was performed at least twice.

FIG. 2.
Promoter architecture of PhhR-controlled genes. Primer extension analysis was used to map transcriptional start sites (Table (Table2).2). Shown are the promoter sequences for four PhhR-dependent transcriptional units: phhA (A), hpd (B), dhcA (C), ...

PhhR purification.

The phhR gene was PCR amplified from P. aeruginosa PA14 chromosomal DNA by using the primers phhR-for-NdeI (5′-GGAATTCCATATGCGTATCAAAGTGCACTGCCAG-3′) and phhR-rev-XhoI (5′-CCGCTCGAGTCAGCCCTCGCCTTGCCCCAC-3′). The resulting 1,560-bp fragment was digested with NdeI/XhoI and ligated into pET15b (Novagen) to generate pKP501. This construct adds a six-histidine tag (His6) to the N terminus of PhhR. For E. coli transformations, 1% glucose was added to the growth medium to suppress transcription of phhR. The sequence of the pKP501 phhR gene was confirmed at the University of Texas Institute for Cell and Molecular Biology DNA Facility. For overexpression of His6-PhhR, the E. coli host strain BL21(DE3) (Novagen) was used. BL21(DE3) carrying pKP501 was subcultured from LB Miller broth supplemented with 100 mM glucose and ampicillin into fresh LB Miller broth with ampicillin. During exponential growth (OD600 of 0.65 to 0.85), 1 mM isopropyl-β-d-thiogalactopyranoside (IPTG) was added to induce expression of His6-phhR. Cultures were harvested for protein purification after 1 h of incubation with IPTG.

To purify His6-PhhR, cells were pelleted by centrifugation at 10,000 × g for 5 min and resuspended in 3 ml cold buffer A (25 mM potassium phosphate buffer, pH 7.4; 0.5 M NaCl; 5 mM DTT; 20 mM imidazole). Cells were lysed by two passes through a French press at ~20,000 lb/in2 at 4°C, and the resulting lysate was centrifuged at 15,600 × g for 20 min to pellet insoluble material. The supernatant was then passed over a 1-ml His-Trap HP column (GE Healthcare) equilibrated with cold buffer A. The column was washed twice with 3 ml cold buffer A, and protein bound to the column was eluted with 3 ml cold buffer B (25 mM potassium phosphate buffer, pH 7.4; 0.5 M NaCl; 5 mM DTT; 500 mM imidazole). All fractions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie blue staining. Western blotting on nitrocellulose membranes with an alkaline phosphatase-conjugated monoclonal anti-polyhistidine clone His-1 antibody (Sigma) and Western Blue stabilized substrate for alkaline phosphatase (Promega) was used to confirm the presence of His6-PhhR in eluted fractions. Purified His6-PhhR was desalted with an Amicon Ultra-4 centrifugal filter device (10-kDa cutoff) by successive concentration and dilution in storage buffer (50 mM Tris-HCl, pH 7.4; 100 mM NaCl; 5 mM DTT; 10% glycerol) until the imidazole concentration was ≤10 mM. Protein concentrations were quantified with the Bio-Rad protein assay (Bio-Rad). Purified His6-PhhR was stored in storage buffer at 4°C and −80°C.

EMSA.

Primers used to generate probes for EMSA are shown in Table Table1.1. Probes were generated by PCR and gel purified. Probes (5 to 10 pmol each) were labeled with [γ-32P]ATP (Sigma-Aldrich), using a KinaseMax kit (Ambion) per the manufacturer's instructions. Unincorporated radiolabeled nucleotides were removed using NucAway spin columns (Ambion). Unlabeled probes targeting intragenic regions of relevant genes were used as cold competitors in EMSAs (see below).

TABLE 1.
Primers used for EMSA

For EMSA, probes (104 cpm) were incubated with various concentrations of His6-PhhR (0, 100, 250, and 500 nM) in 1× DNA binding buffer [20 mM Tris-HCl, pH 7.5; 50 mM KCl; 1 mM EDTA; 1 mM dithiothreitol; 2% glycerol; 100 μg/ml bovine serum albumin; 10 μg/ml poly(dI-dC) (modified from reference 26)]. For each cold competition reaction, a 20 (hpd and dhcA)- or 50 (hmgA and phhA)-fold molar excess of unlabeled probe was added to the binding reaction mix. Competitions were performed with 100 (hpd), 250 (dhcA), or 500 (phhA and hmgA) nM His6-PhhR. The hmgA promoter binding reaction mixture included 100 μM (each) phenylalanine and tyrosine; all other reaction mixtures did not contain free amino acids. EMSA reaction mixtures were incubated at 30°C for 30 min prior to separation in 5% native polyacrylamide gels. Gels were prerun at 80 V for 1 h prior to loading EMSA reaction mixtures. Gels were dried and exposed to phosphorimager screens overnight, and 32P-labeled bands were visualized with a personal molecular imager (Bio-Rad) or Storm 860 imaging system (GE Healthcare Life Sciences).

Microarray data accession number.

The microarray data have been deposited in the EMBL-EBI data bank (www.ebi.ac.uk/miamexpress) under experiment accession number E-MEXP-2593.

RESULTS AND DISCUSSION

Previous work from our laboratory demonstrated that aromatic amino acids within CF sputum not only serve as carbon and energy sources but also enhance synthesis of the cell signaling molecule PQS (19, 21). Although these studies provided new insight into the P. aeruginosa response to nutritional cues, it was not clear if phenylalanine and tyrosine provoke other phenotypic responses. To begin to address this question, the global transcriptional response of P. aeruginosa to aromatic amino acids was assessed using Affymetrix GeneChips. For these experiments, P. aeruginosa was grown in a synthetic CF sputum medium (SCFM) designed to mimic the nutritional conditions of sputum from the CF lung. Twenty-two genes were differentially expressed >4-fold when cells were grown in SCFM compared to SCFM in which the aromatic amino acids had been removed (Table (Table2).2). As expected, genes involved in biosynthesis and response to PQS as well as genes important for catabolism of phenylalanine/tyrosine were highly induced in the presence of aromatic amino acids.

TABLE 2.
Gene expression with aromatic amino acids and in the phhR mutant

In addition to these genes, phhR was also induced in the presence of aromatic amino acids (Table (Table2).2). PhhR has been implicated as a phenylalanine/tyrosine-responsive transcriptional regulator critical for induction of phenylalanine/tyrosine catabolic genes (27). Based on these data, we hypothesized that PhhR was the transcriptional regulator mediating differential expression of the aromatic amino acid-responsive genes observed in Table Table2.2. To test this hypothesis, global expression profiling of wild-type (wt) P. aeruginosa and the isogenic phhR mutant was performed in SCFM. Twelve genes were downregulated >4-fold in the phhR mutant compared to the wild-type strain (Table (Table2).2). All of the genes downregulated in the phhR mutant are putatively involved in phenylalanine/tyrosine degradation to fumarate and acetyl-coenzyme A (acetyl-CoA) (Fig. (Fig.1A)1A) or in amino acid transport. Of note, we propose that DhcA and DhcB constitute the acetoacetyl-CoA transferase (Fig. (Fig.1A),1A), as these proteins share high similarity with the E. coli subunits of this enzyme, namely, AtoD (64% similarity; E value of <10−43 by BLASTp) and AtoA (62% similarity; E value of <10−48 by BLASTp), respectively. Recent observations that dhcA and dhcB likely encode products comprising a 3-dehydrocarnitine-CoA transferase further support this designation (28). Acetoacetate and 3-dehydrocarnitine are structurally similar, with both being 3-ketoacids, differing only by the presence of a trimethylated amine on carbon four of 3-dehydrocarnitine. In addition, isogenic P. aeruginosa dhcA and dhcB mutants displayed reduced growth when phenylalanine was supplied as the major carbon and energy source (data not shown).

FIG. 1.
PhhR is critical for tyrosine/phenylalanine catabolism. (A) Predicted pathway of phenylalanine and tyrosine catabolism in P. aeruginosa (from the Pseudomonas Genome Database [www.pseudomonas.com]). The acetoacetate catabolic pathway is from the Encyclopedia ...

Interestingly, genes involved in PQS biosynthesis were not differentially regulated in the phhR mutant (Table (Table2),2), supporting the hypothesis that while PhhR is likely important for catabolism of phenylalanine/tyrosine, it is not responsible for aromatic amino acid-mediated increases in PQS production. To test this hypothesis, the abilities of aromatic amino acids to support growth and to stimulate PQS production were assessed for wt P. aeruginosa and the phhR mutant. As expected, the P. aeruginosa phhR mutant demonstrated poor growth with phenylalanine and tyrosine as the major carbon and energy source (Fig. (Fig.1B);1B); however growth with tryptophan was unaffected (data not shown). Similar to wt P. aeruginosa, the phhR mutant demonstrated increased PQS production during growth with aromatic amino acids (Fig. (Fig.1C).1C). These results indicate that PhhR is an aromatic amino acid-responsive transcriptional regulator that controls—either directly or indirectly—genes involved in phenylalanine/tyrosine catabolism but not PQS production.

Initial attempts to genetically complement the phhR mutant by a variety of methods, including the use of heterologous inducible promoters, were unsuccessful. We reasoned that this may be due to improper expression of PhhR. To overcome this problem, the phhR Shine-Dalgarno sequence (ribosome binding site) was randomized during PCR amplification by incorporating N5 into the 5′ amplification primer in place of the native phhR Shine-Dalgarno sequence, CGGGC. Amplicons were ligated into the multicopy plasmid pUCP18 and transformed into E. coli. Plasmids were then pooled from the resulting E. coli transformants and transformed into the P. aeruginosa phhR mutant. Plasmids from P. aeruginosa phhR transformants that grew with tyrosine as a carbon source were selected for DNA sequencing. This approach yielded a plasmid (pKP-phhR) containing phhR with a Shine-Dalgarno sequence of GTGCT. The new sequence is likely less favorable to ribosome binding, resulting in tolerable levels of PhhR in the cells. Introduction of pKP-phhR into the P. aeruginosa phhR mutant restored growth with phenylalanine and tyrosine as the primary carbon source (Fig. (Fig.1B1B).

In silico analysis revealed that the PhhR-regulated phenylalanine/tyrosine catabolic genes are arranged into four operons. To identify important transcriptional regulatory DNA elements, promoter regions from these four transcriptional units were mapped using primer extension (Fig. (Fig.22 and Table Table3).3). To map the transcriptional start sites, a nonradioactive primer extension assay previously used to map Helicobacter pylori and P. aeruginosa transcriptional start sites was employed (4, 16). Briefly, a fluorescently labeled primer homologous to the gene of interest was used to generate cDNA from total cellular RNA. The size and quantity of the cDNA product were determined using a standard DNA sequencer and used to map the transcriptional start site of the gene of interest (Table (Table3).3). This analysis revealed that phhA possesses a transcriptional start site located 48 bp upstream of the translational start codon (Fig. (Fig.2A2A and Table Table3),3), hpd possesses a transcriptional start site located 64 bp upstream of the translational start codon (Fig. (Fig.2B2B and Table Table3),3), dhcA possesses a transcriptional start site located 56 bp upstream of the translational start codon (Fig. (Fig.2C2C and Table Table3),3), and hmgA possesses a transcriptional start site located 88 bp upstream of the translational start codon (Fig. (Fig.2D2D and Table Table33).

TABLE 3.
Summary of primer extension data

Promoter regions of phhA, hpd, hmgA, and dhcA were subjected to in silico analyses to identify putative regulatory DNA sequences. A consensus DNA binding site has been proposed for P. putida PhhR (TGTAAAGATAGTTTTACA) (11) and the E. coli PhhR homolog TyrR (TGTAAA-N6-TTTACA) (24). In addition, two potential PhhR binding sites were previously mapped to the phhA-phhR intergenic region of a non-PA14 P. aeruginosa strain (27). Examination of the phhA promoter region in P. aeruginosa PA14 revealed that these two sites are conserved in this strain, centered at −86 and −159 bp respective to the phhA transcriptional start site (Fig. (Fig.2A).2A). Putative PhhR binding sites were also identified upstream of the hpd (−138 bp) (22) and dhcA (−40 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. (Fig.2D2D).

PhhR has been proposed to modulate transcription from σ54- and σ70-dependent promoters (12, 13, 27). Both σ54 and σ70 DNA binding sites have been identified between phhR and phhA on the P. aeruginosa chromosome (27), and PhhA production is σ54 dependent in P. aeruginosa (27). σ54 binding sites in promoters of interest can be difficult to identify in silico. σ54-dependent genes generally possess a conserved GG-N10-GC sequence centered from −24 to −12 bp relative to their transcriptional start sites, although this may vary between promoters (3). For some σ54-dependent promoters, the conserved GG-N10-GC sequence is not centered at positions −24 to −12 but is instead shifted several bases upstream or downstream (3). Of particular note is the algD promoter of P. aeruginosa, which has a GG-N10-GC sequence centered at positions −34 to −22 (31). In addition, while the GG and GC sequences are the most highly conserved sites in the consensus σ54 binding sequence (3), these sites can be variable. Examination of the promoter sequences revealed putative σ54 binding sites upstream of phhA, hpd, and dchA but not hmgA (Fig. 2A to D).

To examine if PhhR binds directly to the promoter regions of PhhR-regulated genes, P. aeruginosa phhR was cloned into an expression vector (pET15b) to create N-terminally His6-tagged PhhR. Affinity purification using a nickel column resulted in nearly pure His6-PhhR, with a prominent band at approximately 58 kDa on Coomassie-stained SDS-PAGE gels (Fig. (Fig.3).3). Binding of His6-PhhR to the phhA, hpd, hmgA, and dhcA promoters was characterized by EMSA. DNA fragments containing promoter regions were generated by PCR (Fig. 4A to D) and 5′-end labeled with 32P for use as EMSA probes. Each probe was incubated with increasing concentrations of PhhR and was submitted to competition with nonradioactive (cold) excess specific and nonspecific competitor DNAs to confirm the specificity of PhhR binding.

FIG. 3.
Purification of His6-PhhR. His6-PhhR was purified using a HisTrap nickel column, separated in a 10% SDS-PAGE gel, and visualized after staining with Coomassie blue. The lanes contained molecular size markers (Std), soluble crude extract (crude), ...
FIG. 4.
His6-PhhR binds to the promoter regions of phenylalanine/tyrosine catabolic genes. (Left) Diagrammatic representation of the phhA (A), hpd (B), dhcA (C), and hmgA (D) promoters. Line arrows indicate transcriptional start sites (Table (Table2), ...

Purified His6-PhhR bound to and retarded mobility of the phhA, hpd, and dhcA promoters in the absence of aromatic amino acids, and the addition of specific, but not nonspecific, cold competitor DNA eliminated this binding (Fig. 4A to C). These results suggest that PhhR is a direct regulator of the phhA, hpd, and dhcA operons. The observation that purified His6-PhhR bound to the phhA, hpd, and dhcA promoters in the absence of aromatic amino acid ligands was not unexpected, as P. putida PhhR binds target promoters in vitro in the absence of amino acid inducers (11). Notably, regulation of the dhcA operon may be more complex, as it was recently reported to also be under the control of the divergently transcribed transcriptional regulator DhcR, although direct binding of this regulator to the dhcA promoter was not examined (28).

We could not detect binding of His6-PhhR to the hmgA promoter (Fig. (Fig.4D),4D), even upon addition of phenylalanine and tyrosine to the binding reaction mix. The P. aeruginosa hmgA promoter does not possess a DNA sequence with high homology to a PhhR binding sequence (Fig. (Fig.2).2). This is in contrast to the hmgA promoter in P. putida, which contains a functional PhhR binding sequence (11). How could P. aeruginosa PhhR regulate hmgA transcription without binding the promoter? Clues were provided by a recent study demonstrating that the transcriptional regulator HmgR also controls hmgA transcription in P. putida (1). HmgR binds and represses transcription of the hmgA promoter unless it is bound to its ligand, homogentisate. Homogentisate is produced during catabolism of phenylalanine/tyrosine by the enzyme Hpd (Fig. (Fig.1A).1A). The P. aeruginosa phhR mutant produced significantly less hpd mRNA (Table (Table2),2), suggesting that this mutant likely produces very little intracellular homogentisate, even in the presence of phenylalanine/tyrosine. The low levels of homogentisate would not allow derepression of HmgR, thereby resulting in lower levels of hmgA mRNA in the phhR mutant. In support of this hypothesis, P. aeruginosa possesses an HmgR gene homolog (PA2010 in strain PAO1 and PA14_38500 in strain PA14) immediately upstream of hmgA, and the hmgA promoter possesses a putative HmgR binding site (1).

The goal of this study was to expand on previous work from our laboratory demonstrating that aromatic amino acids within CF lung secretions (sputum) not only serve as carbon and energy sources but also enhance PQS synthesis. This study provides evidence that PhhR controls genes important for catabolism of phenylalanine and tyrosine. The role of PhhR in regulation of phenylalanine catabolic genes is reminiscent of P. putida PhhR (11); however, it is intriguing that in contrast to P. putida PhhR, P. aeruginosa PhhR does not control expression of aromatic amino acid biosynthesis genes (11). This study also provides evidence that PhhR is not critical for induction of PQS biosynthetic genes. Instead, the data support a previous model of enhanced PQS production in which the presence of phenylalanine/tyrosine allows increased flux of the common biosynthetic precursor chorismate away from aromatic amino acid production and into PQS biosynthesis (19).

Acknowledgments

This work was funded by a grant from the NIH (grant 5R01AI075068 to M.W.). M.W. is a Burroughs Wellcome Investigator in the Pathogenesis of Infectious Disease.

We thank Holly K. Huse and Ryan Mak for preparing RNA for GeneChip analysis.

Footnotes

[down-pointing small open triangle]Published ahead of print on 19 March 2010.

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