Molecular Characterization of PauR and Its Role in Control ofPutrescine and Cadaverine Catabolism through the �-GlutamylationPathway in Pseudomonas aeruginosa PAO1

Han Ting Chou,a Jeng-Yi Li,a Yu-Chih Peng,a Chung-Dar Lua,b

Department of Biology, Georgia State University, Atlanta, Georgia, USAa; Department of Medical Laboratory Sciences and Biotechnology, China Medical University,Taichung, Taiwanb

Pseudomonas aeruginosa PAO1 grows on a variety of polyamines as the sole source of carbon and nitrogen. Catabolism of poly-amines is mediated by the �-glutamylation pathway, which is complicated by the existence of multiple homologous enzymeswith redundant specificities toward different polyamines for a more diverse metabolic capacity in this organism. Through a se-ries of markerless gene knockout mutants and complementation tests, specific combinations of pauABCD (polyamine utiliza-tion) genes were deciphered for catabolism of different polyamines. Among six pauA genes, expression of pauA1, pauA2, pauA4,and pauA5 was found to be inducible by diamines putrescine (PUT) and cadaverine (CAD) but not by diaminopropane. Activa-tion of these promoters was regulated by the PauR repressor, as evidenced by constitutively active promoters in the pauR mu-tant. The activities of these promoters were further enhanced by exogenous PUT or CAD in the mutant devoid of all six pauAgenes. The recombinant PauR protein with a hexahistidine tag at its N terminus was purified, and specific bindings of PauR tothe promoter regions of most pau operons were demonstrated by electromobility shift assays. Potential interactions of PUT andCAD with PauR were also suggested by chemical cross-linkage analysis with glutaraldehyde. In comparison, growth on PUT wasmore proficient than that on CAD, and this observed growth phenotype was reflected in a strong catabolite repression of pauApromoter activation by CAD but was completely absent as reflected by activation by PUT. In summary, this study clearly estab-lishes the function of PauR in control of pau promoters in response to PUT and CAD for their catabolism through the �-glu-tamylation pathway.

Biogenic polyamines are essential for cell growth and partici-pate in a wide spectrum of physiological functions in living

organisms (1, 2). Common compounds in this group include thediamines 1,3-diaminopropane (DAP), putrescine (PUT), and ca-daverine (CAD), the triamines spermidine (SPD) and norspermi-dine, and the tetramine spermine (SPM). In microbes, PUT andCAD are two most common biogenic diamines. Intracellular con-centrations of these compounds are finely tuned through biosyn-thesis, degradation, and transport (3). PUT and CAD are synthe-sized directly from decarboxylation of ornithine (speC) and lysine(ldcA), respectively (4, 5). Alternatively, PUT can also be gener-ated through arginine and agmatine degradation (6, 7). On theother hand, exogenous PUT and CAD can be taken as carbon andnitrogen sources by many bacteria, including Pseudomonasaeruginosa (4, 7, 8). It was first reported for Escherichia coli thatPUT is degraded through the �-glutamylation pathway (9, 10).This pathway consists of four consecutive reactions with con-sumption of ATP in the first step for �-glutamylputrescine syn-thesis (Fig. 1A), and one single set of puuABCD genes encodesthese four enzymes in the pathway.

The �-glutamylation pathway is further expanded in Pseu-domonas to accommodate a more diverse metabolic capacity.Different from that in E. coli, the �-glutamylation pathway ismore complex because of the existence of multiple homolo-gous enzymes with redundant specificities toward differentpolyamines for a larger metabolic capacity in P. aeruginosa(11). Through transcriptome analysis, we reported identifica-tion of a set of redundant pauABCD genes that are essential forpolyamine utilization via the �-glutamylation pathway in P.aeruginosa (7, 11). These genes include six puuA (pauA1 to

pauA6), four puuB (pauB1 to pauB4), one puuC (pauC), andtwo puuD (pauD1 and pauD2) homologues. From growth phe-notype analysis of a series of unmarked pauA mutants, specificcombinations of pauA genes were assigned to catabolism ofDAP, PUT, CAD, and SPM (11).

Expression of puuABCD genes in E. coli is subjected to regula-tion by the PuuR repressor (10). Due to the redundancy of paugenes for a more diverse scope of substrates, regulation of poly-amine catabolism in P. aeruginosa was expected to be more com-plicated than that in E. coli. The BauR protein was identified as atranscriptional activator of the pauA3B2 operon for DAP catabo-lism. At least three other regulatory genes were located in physicalproximity to the scattered pau genes. Based on sequence compar-ison, the PA5301 gene of P. aeruginosa was proposed to encode aputative homologue of E. coli PuuR.

This study was undertaken to further decipher the geneticcombinations of all pauABCD genes for the catabolism of poly-amines. Expression of four PUT- and CAD-dependent pauA pro-moters was subjected to detailed analysis to elucidate the inducersignal molecules of these promoters. Genetic and biochemicalcharacterization of PA5301 and its encoded PauR protein estab-lished their roles in control of PUT- and CAD-responsive pauA

Received 7 March 2013 Accepted 19 June 2013

Published ahead of print 21 June 2013

Address correspondence to Chung-Dar Lu, biocdl@gsu.edu.

Copyright © 2013, American Society for Microbiology. All Rights Reserved.

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promoters, independent of BauR in control of DAP-responsivegenes.

MATERIALS AND METHODSBacterial strains, plasmids, media, and growth conditions. Luria-Ber-tani (LB) medium was used with the following supplements as required:ampicillin at 100 �g ml�1, tetracycline at 12.5 �g ml�1, gentamicin at 10�g ml�1, and 5-bromo-4-chloro-3-indolyl-�-D-galactopyranoside (X-Gal) at 0.03% (wt/vol) for E. coli and carbenicillin at 100 �g ml�1, strep-tomycin at 500 �g ml�1, tetracycline at 100 �g ml�1, and gentamicin at100 �g ml�1 for P. aeruginosa. Minimal medium P (MMP) (12) contain-ing carbon sources at 20 mM and nitrogen sources at 5 mM was used forthe growth of P. aeruginosa.

Cloning of pauB, pauD, and spuC genes for complementation tests.In general, each gene was amplified by PCR with a pair of primers thatcovers the entire sequence of its structural gene and the ribosomalbinding site. The PCR products were cloned into appropriate restric-tion sites on pUCP18 so that expression of the cloned gene was drivenby the upstream lac promoter. Positive clones were first identified in E.coli and confirmed by sequencing, followed by transformation intostrains of P. aeruginosa.

Construction of knockout mutants. For the pauR mutant, two flank-ing regions of the targeted gene were PCR amplified and cloned intopRTP1 using primers pauR1F (5=-CGG GAT CCC GAT CAG AAA TTTGCG GGC GTG G-3=), pauR1R (5=-CGG AAT TCC GGC GCG GTGTCC ATG CGC CTG-3=), pauR2F (5=-CGG AAT TCC GGT CGA TCAGTT CGT CTG CCT-3=), and pauR2R (5=-CCA AGC TTG GGG TGCTCG TCT GCA AAC CAT-3=). The PCR products were cloned intopRTP1. A cassette carrying a gentamicin resistance gene from pGM�1was inserted into the conjunction of the two DNA fragments (13). Forgene replacement, E. coli SM10 served as the donor in biparental matingwith PAO1-Smr (14). The desired knockout mutants were selected on LBplates containing streptomycin and either gentamicin or tetracycline, andthe mutation was confirmed by PCR. For the construction of a series ofpauB and pauD mutants, the protocol for gene replacement and in vivoexcision by the Flp-FRT recombination system (15) was used to generateunmarked mutants of PAO1. Expected deletions in these mutants were

confirmed by PCR; the locations of the deleted regions in each gene aredescribed in Table 1.

Construction of lacZ fusions. Plasmids pGU101 and pGU102 wereused to measure pauA2 and pauA1 promoter activities, respectively (7,16). Regulatory regions of pauA4 (PA2040) and pauA5 (PA3356) wereamplified by PCR from the genomic DNA of P. aeruginosa PAO1 using thefollowing primers: PpauA5F (5=-GGT GGA TCC GAG AAT CAA CGGCAG TAC TC-3=), PpauA5R (5=-GGT AAG CTT GAT GCA TTG CAGCAG CAC GCCA-3=), PpauA4F (5=-GGT GGA TCC TTG AAC GAT CTTGCT CTT CGT ATC-3=), and PpauA4R (5=-GGT AAG CTT GAA GGAACA GGC TCG GCT CAG-3=). PCR products were cloned into pQF50and confirmed by DNA sequencing.

Measurements of LacZ enzyme activity. The cells were grown inMMP containing carbon and nitrogen sources as indicated below. Cells inthe mid-log phase when the optical density at 600 nm reached 0.7 wereharvested by centrifugation and then passed through a French press at8,500 lb/in2. The cell debris were removed by centrifugation at 20,000 � gfor 10 min at 4°C, and protein concentrations in the crude extracts weredetermined by the Bradford method (17) using bovine serum albumin asa standard. The levels of �-galactosidase activity were measured at 37°Cusing o-nitrophenyl-�-galactopyranoside as the reaction substrate, andthe formation of o-nitrophenol was determined by spectrophotometry at420 nm (18).

Overexpression and purification of the histidine-tagged PauR pro-tein. Full-length pauR was amplified from the genomic DNA of P.aeruginosa using the following primers: pauRF (5=-GAC GTC GGTGCT CGT CTG CAA ACC-3=) and pauRR (5=-GAG GAA TTC TTGTGC TTC AGG TCA GAA ATT TGC-3=). The PCR products weredigested with EcoRI restriction endonuclease and assembled onto theSmaI and EcoRI sites of pBAD-His6, a modified pBAD expressionvector (19, 20). For overexpression, Escherichia coli TOP10 harboringthe recombinant plasmid was grown in LB medium containing ampi-cillin at 37°C until the optical density at 600 nm reached 0.5, at whichpoint 0.2% arabinose was added to the culture for induction. Culturegrowth was continued for another 4 h under the same conditionsbefore harvest by centrifugation. A cell extract was obtained by passing

FIG 1 �-Glutamylation pathway for polyamine catabolism and schematic presentation of pau loci in P. aeruginosa. (A) The pathway shown is representative fordiamines only. The n values of DAP, PUT, and CAD are 3, 4, and 5, respectively, and these three diamines are converted into �-alanine, �-aminobutyrate, and�-aminovalerate through this pathway. (B) Gene organizations of loci containing pauA1 to pauA6, pauB1 to pauB4, pauC, and pauD1 and pauD2 for polyamineutilization. Genes in these loci are presented as horizontal arrows and labeled with the corresponding PA gene numbers according to the PAO1 genomeannotation. Putative functions of uncharacterized and hypothetical proteins are in parentheses. Promoters under PauR regulation as analyzed in this study areindicated with stars.

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through a French press cell at 8,500 lb/in2. The soluble fraction wassubjected to protein purification using a HisTrap HP column (GEHealthcare) by following the manufacturer’s instructions. Protein pu-rity was analyzed by SDS-PAGE, and protein concentration was deter-mined by the method of Bradford (17).

Glutaraldehyde cross-linking. Purified His-PauR was subjected toglutaraldehyde treatment in 20 mM HEPES buffer at pH 7.5. Reactionmixtures with 25 �g of purified His-PauR in a total volume of 100 �l weretreated with 5 �l of 2.3% freshly prepared solution of glutaraldehyde for 2min at 25°C followed by 3 min at 37°C. The reaction was terminated byaddition of 10 �l of 1 M Tris-HCl (pH 8.0). Diamines, �-aminobutyrate(GABA), or �-aminovalerate (AMV) was incorporated into the reactionmixture at 1 mM concentration either prior to glutaraldehyde treatmentor after quenching with Tris buffer. Cross-linked proteins were analyzedby SDS-PAGE and visualized by ProtoBlue Safe staining (National Diag-nostics).

Electrophoretic mobility shift assays. For the regulatory regions ofthe pau operons, DNA fragments covering the entire intergenic re-gions as defined by the genome annotation website www.pseudomonas.com were PCR amplified from the genome by PCR withprimers. For radioactively labeled DNA probes, [�-32P]dATP was in-corporated by polynucleotide kinase. DNA probes (0.2 ng) were al-lowed to interact with different concentrations of purified proteins ina 20-�l reaction mixture containing 50 mM Tris-HCl (pH 8), 50 mMKCl, 1 mM EDTA, 5% (vol/vol) glycerol, and 150 �g/ml of acetylatedbovine serum albumin. A smaller DNA fragment amplified from thelipA promoter region was used as a negative control for nonspecificbinding. The reaction mixtures were incubated for 30 min at roomtemperature before being applied to a 4% native polyacrylamide gel in0.5� Tris-borate-EDTA (TBE) (pH 7.5) buffer. After being dried, thegel was autoradiographed by exposure to a phosphorimager plate,scanned using FLA-7000, version 1.1, and analyzed by Multi Gauge,

TABLE 1 Strains and plasmids used in this study

Strain or plasmid Genotype or descriptiona Source or reference

E. coli strainsDH5� F� 80dlacM15 (lacZYA-argF)U169 deoR recA1 endA1 hsdR17 (rK

� mK�) supE44

�� thi-1 gyrA96 relABethesda Research

LaboratoriesTOP10 F� mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 araD139 (ara-leu)7697

galU galK rpsL (Smr) endA1 nupGInvitrogen

SM10 thi-1 thr leu tonA lacY supE recA::RP4-2-Tc::Mu (Kmr) 22

P. aeruginosa strainsPAO1 Wild type 12PAO1-Smr Spontaneous Smr mutant strain of PAO1 23PAO5722 pauR::Gmr This studyPAO5725 pauA1A2A3A4A5A6 11PAO5008 spuC::Tcr 16PAO5730 pauB1B2B3B4; spuC::Tcr This studyPAO5731 pauB1; from Ser-63 to Arg-362 This studyPAO5732 pauB2; from His-6 to Leu-413 This studyPAO5733 pauB3; from Lys-123 to Arg-318 This studyPAO5734 pauB4; from Asn-139 to Leu-331 This studyPAO5735 pauB1B2B3B4 This studyPAO5707 pauC::Gmr (kauB::Gmr) 7PAO5737 pauD1; from Leu-38 to Cys-243 This studyPAO5738 pauD2; from Arg-50 to Thr-223 This studyPAO5739 pauD1D2 This study

PlasmidspRTP1 Ampr Sms conjugation vector 24pGM�1 Ampr Gmr; gentamicin resistance gene cassette with omega loop on both ends 13pQF50 bla; lacZ transcriptional fusion vector 25pQF52 bla; lacZ translational fusion vector derived from pQF50 26pGU101 PpauA2::lacZ (spuA) translational fusion of pQF52 16pGU102 PpauA1(spuI)::lacZ translational fusion of pQF52 16pHT2040 PpauA4::lacZ transcriptional fusion of pQF50 This studypHT3356 PpauA5::lacZ transcriptional fusion of pQF50 This studypBAD-HisD Modified protein expression vector derived from pBAD-HisA 19pBAD-PauR 6� His-tagged PauR protein expression vector This studypPAUB1 pauB1 (PA0534) in pUCP18 This studypPAUB2 pauB2 (PA1565) in pUCP18 This studypPAUB3 pauB3 (PA2776) in pUCP18 This studypPAUB4 pauB4 (PA5309) in pUCP18 This studypSPUC spuC (PA0299) in pUCP18 This studypPAUD1 pauD1 (PA0297) in pUCP18 This studypPAUD2 pauD2 (PA1742) in pUCP18 This studypUCP18 E. coli/P. aeruginosa shuttle vector 27

a Smr, streptomycin resistance; Kmr, kanamycin resistance; Gmr, gentamicin resistance; Tcr, tetracycline resistance; Ampr, ampicillin resistance; Sms, streptomycin sensitive.

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version 3.0 (Fujifilm). For nonradioactive DNA probes, 1 ng of DNAwas used in the reaction mixture and visualized from the polyacryl-amide gel by SYBR green I staining (Invitrogen) followed by an OmegaUltraLum imaging system with 473-nm excitation and 520-nm emis-sion wavelengths.

RESULTS AND DISCUSSIONGene knockout analyses decipher specific combinations ofpauABCD genes for polyamine catabolism. As shown in Fig. 1A,diamine catabolism through the �-glutamylation pathway re-quires four steps of enzymatic reactions, which serve to convertDAP, PUT, and CAD into �-alanine, GABA, and AMV, respec-tively. As evidenced by genetic studies and transcriptome analysis(11, 21), multiple enzymes of redundant substrate specificitieswere proposed to participate in this pathway of polyamine catab-olism—six PauA, four PauB, one PauC, and two PauD enzymes.We have reported that PauA3 is essential for DAP catabolism,PauA1A2A4 for PUT, PauA1A4A5 for CAD, and PauA2 for tri-amine SPD and tetramine SPM (11). PauC is the only enzyme ofbroad substrate specificity catalyzing the third step in the pathway,and the pauC mutant cannot grow on all polyamines mentionedabove.

To further differentiate the functions of PauB and PauDenzymes in catabolism of these polyamines, a series of pauBand pauD knockout mutants were constructed and subjected togrowth phenotype analysis. The results in Table 2 indicate thata single pauB1 lesion caused partial growth retardation onCAD, pauB2 mutation abolished DAP utilization, and thepauB4 mutant cannot grow on SPD and SPM. Surprisingly,strain PAO5731 devoid of four pauB genes did not exhibit anyapparent growth defect on PUT, and its growth on CAD wascomparable to that of the pauB1 mutant. These results indi-

cated the presence of an additional enzyme(s) for polyaminedeamination.

The PauB enzymes were proposed to catalyze FAD-depen-dent oxidative deamination of �-glutamylpolyamine. BesidesPauB enzymes, genetic analysis from our previous studies (7,16) supported participation of a putative putrescine:pyruvatetransaminase SpuC in PUT catabolism. As shown in Table 2,while PAO5008 with a lesion on spuC was partially defective ingrowth on PUT or CAD as the sole source of carbon or nitrogenin the liquid medium, growth on these two diamines was com-pletely abolished in PAO5730 devoid of spuC and all four pauBgenes. The growth phenotypes of these three mutant strainswere also analyzed on agar plates. As shown in Fig. 2, the resultswere consistent with those in the liquid medium (Table 2) ex-cept for PAO5730 on PUT, which exhibited growth of distinctcolonies derived from the inoculation spot. It is very likely thatthese outgrowth colonies were from spontaneous suppressorsof PAO5730 when exposed to PUT; however, the nature of thisspecific stress was not clear.

Complementation tests were conducted to further characterizethe physiological functions of spuC and pauB genes on polyamineutilization. These genes were cloned individually into pUCP18and introduced into PAO5730. The recombinant strains ofPAO5730 were subjected to growth phenotype analysis on poly-amines. As shown in Table 2, growth of PAO5730 on DAP canonly be recovered by the pauB2 or spuC clone, growth on PUT wasrecovered by pauB1, pauB3, or spuC, and growth on CAD wasrecovered by pauB1 or spuC. Surprisingly, none of the pauB andspuC genes can make PAO5730 to regain growth on SPD andSPM. However, PAO5374 (pauB4) harboring pPAUB4 indeedrestored growth on SPD and SPM. From these results, we con-cluded that the PauB4 enzyme may catalyze the reaction to breakthe CON bond of the internal secondary amine of SPD and SPMto split it into entities with either a three-carbon or four-carbonchain length, which can be further degraded by the same route aseither DAP or PUT.

For the pauD1 and pauD2 genes, the results in Table 2 demon-

TABLE 2 Growth phenotypes of pauB, pauD, and spuC mutants onpolyamines

Strain Genotype

Growth responsea in MMPcontaining:

Glu DAP PUT CAD SPD

PAO1 Wild type � � � � �PAO5722 pauR � � � � �PAO5731 pauB1 � � � �PAO5732 pauB2 � � � � �PAO5733 pauB3 � � � � �PAO5734 pauB4 � � � � �PAO5735 pauB1B2B3B4 � � � �PAO5008 spuC � � �PAO5730 pauB1B2B3B4 spuC � � � � �PAO5370/pSPUC pauB1B2B3B4

spuC/spuC�� � � � �

PAO5730/pPAUB1 pauB1B2B3B4spuC/pauB1�

� � � � �

PAO5730/pPAUB2 pauB1B2B3B4spuC/pauB2�

� � � � �

PAO5730/pPAUB3 pauB1B2B3B4spuC/pauB3�

� � � � �

PAO5730/pPAUB4 pauB1B2B3B4spuC/pauB4�

� � � � �

PAO5734/pPAUB4 pauB4/pauB4� � � � � �PAO5737 pauD1 � � � � �PAO5738 pauD2 � � � � �PAO5739 pauD1D2 � � � � �PAO5739/pPAUD1 pauD1D2/pauD1� � � � � PAO5739/pPAUD2 pauD1D2/pauD2� � � � � �a �, growth in 24 h; , growth in 48 h; �, no growth in 48 h.

FIG 2 Growth phenotype analysis of pau mutants on agar plates. Aliquots ofcell suspension with comparable numbers of cells were prepared as describedin Materials and Methods and spotted on the minimal medium plates with theindicated compounds (10 mM) as the sole sources of carbon and nitrogen.Strains are indicated as follows: WT, wild-type PAO1; pauB, PA5735 devoid ofall four pauB genes; spuC, PA5008; pauB spuC, PA5730 devoid of spuC and allfour pauB genes. Glu, L-glutamate; Put, putrescine; Cad, cadaverine; Spd, sper-midine.

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strate that pauD2 is essential for growth on diamines DAP, PUT,and CAD. While growth on SPD or SPM was sustained in mutantswith a single knockout in pauD1 or pauD2, no growth on thesetwo compounds can be observed in the strain with pauD1 andpauD2 double lesions. In the �-glutamylation pathway for di-amines, PauD1 and PauD2 were proposed to hydrolyze the final�-glutamylated intermediates to release and ensure recycle of glu-tamate moiety. The results of complementation tests of thepauD1D2 double mutant with pPAUD1 and pPAUD2 (Table 2)support the proposed functions of pauD1 and pauD2 on poly-amine utilization, with pauD2 playing a more important rolethan pauD1. These results were also consistent with the currentmodel of SPD and SPM degradation in which �-glutamylationmay take place on either of the two terminal primary amines,followed by the cleavage of the secondary amine. While morework is needed to elucidate details from this point on, �-glu-tamyl-GABA and �-glutamyl-�-alanine are two apparentproducts of this catabolic pathway, and therefore only thepauD1D2 double mutant was able to block completely SPDand SPM catabolism in this pathway.

Through the exhaustive genetic analyses reported here, wewere able to decipher specific combinations of genes for catabo-lism of different diamines via the �-glutamylation pathway in P.aeruginosa: pauA3-pauB2-pauC-pauD2 for DAP, pauA1A2A4-pauB1B3 (SpuC)-pauC-pauD2 for PUT, and pauA1A4A5-pauB1(SpuC)-pauC-pauD2 for CAD. In addition, since PUT in excesscan be channeled into SPD through the SPD biosynthetic path-way, it may therefore provide another route for PUT utilizationafter conversion into SPD.

PauR effect on pauA promoters. The five pauA genes (pauA1to pauA5) involved in diamine catabolism are expressed fromfive independent transcriptional units (Fig. 1B). The BauR pro-tein, a transcriptional regulator of the LysR family, is requiredfor DAP- and SPD-dependent induction of the pauA3 pro-moter (11), and the bauR mutant affects growth on DAP and

SPD but not on CAD and PUT. Therefore, we hypothesizedthat four PUT- and CAD-related pauA genes (pauA1A2A4A5)are subjected to a regulatory mechanism different from BauRon pauA3. One promising candidate gene was PA5301, whichencodes a transcriptional regulator exhibiting 44% sequenceidentity to E. coli PuuR for putrescine utilization (10). As de-scribed below, we provided several lines of evidence to supportPA5301 in regulation of polyamine utilization, and hencePA5301 is designated pauR from now on.

Strain PAO5722, a pauR knockout mutant, was constructed asdescribed in Materials and Methods and subjected to growth phe-notype analysis for polyamine utilization. As shown in Table 2, noapparent growth defect was observed in this mutant on any of thepolyamines tested (DAP, PUT, CAD, and SPD). Although thisresult indicated that PauR is not essential for pau gene expression,we proposed that PauR may serve as a transcriptional repressor forpau operons. To test this hypothesis, the expression patterns ofpauA promoters fused to the lacZ reporter gene on recombinantplasmids were determined by measurements of �-galactosidaseactivities in the wild-type strain PAO1 and the pauR mutant. Thecells were grown in glucose minimal medium with either ammo-nium or polyamines as the nitrogen source. As shown in Fig. 3, thepromoters of pauA1, -A2, -A4, and -A5 were specifically inducedby PUT and CAD in the wild-type strain PAO1. When introducedinto the pauR mutant, the basal levels of these promoters in-creased significantly, and polyamine-dependent induction ofthese promoters was diminished in this mutant. In comparison,the pauA3 promoter was specifically activated by DAP in PAO1(11), and the expression pattern of this promoter remained thesame in the pauR mutant (data not shown). These results pro-vided the first line of evidence for PauR as a transcriptional repres-sor of the pauA1, -A2, -A4, and -A5 promoters but not the pauA3promoter (11).

Enhanced PUT- and CAD-dependent induction of the pauApromoters in the mutant devoid of PauA enzymes. To test if

FIG 3 Effect of pauR on expression profiles of the pauA promoters. Specific activities of �-galactosidase expressed from pauA promoter-lacZ fusions weremeasured from the host strains wild-type PAO1 (solid bars) and pauR mutant PAO5722 (empty bars). The cells were grown in minimal medium P supplementedwith glucose (G) as the sole source of carbon and putrescine (P), cadaverine (C), or ammonia (N) as the sole source of nitrogen.

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PUT and CAD are inducers of the pauA promoters, the activi-ties of these promoters from recombinant plasmids as de-scribed above in response to exogenous PUT and CAD werecompared in the wild-type strain PAO1 and strain PAO5725, inwhich six pauA genes were deleted. Without functional PauAenzymes, PAO5725 was expected to accumulate PUT or CADsupplement inside the cells to levels higher than that in PAO1,and synthesis of the downstream intermediate compounds inthe �-glutamylation pathway would be blocked in this mutant.As shown in Fig. 4, the promoters of pauA1, pauA2, pauA4, andpauA5 were inducible by either PUT (�5-fold) or CAD (�2.5-fold) in PAO1 grown in the glucose-ammonia minimal me-dium. In PAO5725, while the basal expression levels of thesefour pauA promoters were comparable to those in PAO1, thefold induction by PUT and CAD for the pauA1, pauA2,and pauA4 promoters was further enhanced significantly inPAO5725. These data support PUT and CAD as the inducermolecules of the corresponding pauA promoters.

In contrast, we have reported that DAP-dependent induc-tion of the pauA3 promoter was greatly diminished in the mu-tant without functional PauA enzymes, and that �-alanine de-rived from DAP through the �-glutamylation pathway mayserve as the signal molecule for the BauR-dependent regulatorycircuit (11).

Specific interactions of PauR with the promoter regions ofpauABCD genes in vitro. To further support PauR as a tran-scriptional repressor of pauA promoters, PauR with a hexahis-tidine tag at its N terminus was constructed and purified tohomogeneity as described in Materials and Methods. TheDNA-binding activity of this recombinant PauR protein wassubjected to analysis by electrophoretic mobility shift experi-ments using the same regulatory regions of the four pauA pro-moter fusions in expression measurements. As shown in Fig. 5,the presence of nucleoprotein complexes with a retarded mo-bility clearly demonstrated binding of PauR specifically tothese pauA promoter regions with high affinity.

Potential interactions of PauR with the promoter regions ofother pauABCD genes (Fig. 1B) were also tested. As shown inFig. 5, PauR binds to the regulatory regions of all pau operonsexcept pauA3B2 and pauB1. We have reported that expressionof the pauA3B2 operon is regulated by BauR in control of DAPand �-alanine utilization (11). In the case of pauB1, it might besubjected to regulation by a putative transcriptional regulatorencoded by the gene immediately downstream of pauB1.

In the current hypothesis, the repression effect of PauR on theaffected promoters would be released upon its interactions withthe inducer molecule PUT or CAD. Interactions of PauR with thepromoter regions were also tested with inclusion of PUT or CADin the reactions. However, no apparent effect was detectable onthe DNA-binding activity of PauR with either 1 mM or 5 mMdiamines in the reactions (data not shown). We did not continuethe experiments with higher concentrations of polyamines, as itwas well known that polyamines can bind to DNA through chargeinteractions and therefore may potentially interfere with PauRactivity nonspecifically.

Cross-linkage analysis revealed potential interactions ofPauR with PUT and CAD. Cross-linking analysis with glutaralde-hyde was also conducted to detect any conformational changes ofPauR in response to polyamines. As shown in Fig. 6, an additionaldistinct peptide band corresponding to the dimeric PauR forma-tion was detected after cross-linking. When PUT or CAD wasadded into the reaction, the peptide bands for monomeric anddimeric PauR became diffused, with a slight increase in molecularweight, on SDS-PAGE. This change is specific to PUT and CADbut not to DAP, a diamine with a shorter methylene chain. In thecontrol group, polyamines added after completion of cross-link-ing reactions did not generate detectable band diffusion. Thesedata supported PauR interaction specifically with PUT and CADbut not DAP.

Catabolite repression on CAD-dependent induction of pauApromoters. Although PUT- and CAD-dependent regulation ofpauA expression involved PauR, induction of these pauA promot-

FIG 4 Expression profile of pauA promoters in P. aeruginosa PAO1. Specific activities of �-galactosidase, expressed from pGU102 for the pauA1 promoter,pGU101 for the pauA2 promoter, pHT2040 for the pauA4 promoter, or pHT3356 for the pauA5 promoter, were measured from the host strains wild-type PAO1(solid bars) and pauA1A2A3A4A56 mutant PAO5725 (empty bars) devoid of six glutamyl-polyamine synthetase genes. The cells were grown in glucose-ammonia (GN) minimal medium in the presence or absence of putrescine (P) and cadaverine (C).

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ers by PUT or CAD responded differently to other nutrientsources. As shown in Fig. 7, PUT-dependent induction of pauApromoters in wild-type PAO1 was not affected by the addition ofglucose and ammonia to the minimal medium. In comparison,CAD-dependent induction of all these four pauA promoters wassignificantly suppressed by exogenous glucose and ammonia.Similar patterns of repression by exogenous glutamate were also

observed (data not shown). The molecular nature of this repres-sion effect was not clear. It might be mediated through differentialeffects on PUT-PauR and CAD-PauR complexes or on uptake ofexogenous CAD and PUT.

In summary, specific genetic combinations of pauABCD genesfor catabolism of different polyamines were deciphered in thisstudy. The PauR repressor was characterized as a transcriptionalregulator in response to PUT- and CAD-dependent induction ofpauA1, -A2, -A4, and -A5 promoters. The PauR protein was able

FIG 5 PauR regulation on pau promoters. Interactions of purified His-PauR with pau promoters were demonstrated by electrophoretic mobility shift assays asdescribed in Materials and Methods. Ctrl, negative control DNA.

FIG 6 PauR conformational change in response to putrescine and cadav-erine. PauR showed distinct dimer formation after glutaraldehyde cross-linkage (CL). The presence of PUT or CAD, but not DAP, in the reactionsgenerated more diffused cross-linking products. In the control group,polyamines were added after the termination of cross-linking reactions.Reactions including polyamines but no glutaraldehyde are also shown as anegative control.

FIG 7 Glucose-ammonia effect on expression profile of pauA promoters.Specific activities of �-galactosidase expressed from pauA promoter-lacZ fu-sions were measured from the host strain (wild-type PAO1). The cells weregrown in minimal medium P in the presence of putrescine (Put) or cadaverine(Cad). Empty bars represent expression levels when 10 mM putrescine orcadaverine was used as the sole source of nutrient for growth. Solid bars showrepression on cadaverine utilization by 10 mM glucose and 5 mM ammonia asbackground nutrients, while pauA expression with putrescine remained unaf-fected.

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to bind to the regulatory regions of all pau operons exceptpauA3B2 and pauB1. In conjunction with our previous report ofBauR on the pauA3 promoter for DAP catabolism (11), we estab-lished a clear regulatory circuit by PauR and BauR in control ofdiamines catabolism. Further studies are in progress to elucidatethe regulatory mechanism of pauABCD genes in response to SPDand SPM.

ACKNOWLEDGMENTS

This work was supported in part by National Science Foundation(MCB0950217) to C.-D. Lu and by the Molecular Basis of Disease Pro-gram fellowship of the Georgia State University to H. T. Chou and J.-Y. Li.

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