Pyoverdine-Mediated Regulation of FpvA Synthesis in Pseudomonas aeruginosa: Involvement of a Probable Extracytoplasmic-Function Sigma Factor, FpvI

A search of the pvd pyoverdine biosynthesis locus of Pseudomonasaeruginosa identified an open reading frame, PA2387, whose productexhibited a sequence similar to those of a number of so-calledextracytoplasmic- function sigma factors responsible for siderophore-dependentexpression of iron-siderophore receptors in Escherichia coliand Pseudomonas putida. Deletion of this gene, dubbed fpvI,compromised pyoverdine-dependent FpvA ferric pyoverdine receptorproduction and fpvA gene expression, while the cloned gene stimulatedfpvA expression. A Fur-binding site was identified immediatelyupstream of fpvI, consistent with the observed iron-regulatedexpression of fpvI and fpvA.

INTRODUCTION

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Introduction

With few exceptions, almost all bacteria require iron for growthand survival (27). Iron acquisition in nature is complicated,however, due to the low solubility of iron under aerobic conditionsat neutral pH (27). Pathogenic organisms face similar restrictionsin human hosts, since iron is generally sequestered intracellularlyin heme-containing compounds, or in fluids, by iron-bindingproteins such as lactoferrin and transferrin (39). Many bacteriaovercome this problem by synthesizing high-affinity iron chelatorscalled siderophores (29). Together with siderophore-specificouter membrane receptors, these facilitate the uptake of ironrequired to sustain growth and pathogenesis (28).

Pseudomonas aeruginosa, an opportunistic human pathogen (10),produces two known siderophores, pyoverdine (6) and pyochelin(5), in response to iron limitation. Pyoverdine is the superiorchelator, at least at neutral pH (23), and is required for invivo growth and virulence (24, 34, 48). Genes involved in thebiosynthesis of pyoverdine localize in two gene clusters, thepvc operon (45, 46) and the pvd locus (22, 26, 36, 50), whichare implicated in the synthesis of the chromophore and peptidemoieties, respectively. The fpvA gene encoding the ferric pyoverdinereceptor is also localized in the pvd cluster (22, 35, 36).

Although it is an essential nutrient for growth and pathogenesis,iron, in excess, is toxic to cells (12). Thus, uptake genesare tightly regulated by intracellular iron levels, mediatedby the Fur repressor (8, 13, 38). Though a Fur homologue hasbeen identified in P. aeruginosa (38), it does not directlyregulate genes involved in pyoverdine biosynthesis. Rather,an alternative sigma factor, PvdS (7, 21, 26, 51), which positivelyregulates the expression of several pyoverdine biosyntheticgenes (20, 52), is itself regulated by Fur (7, 20, 26). Pyoverdinecontrol of FpvA expression has also been reported (11, 35),reminiscent of siderophore-dependent receptor gene expressionin Escherichia coli (14) and Pseudomonas putida (17). In E.coli, ferric dicitrate upregulates its receptor, FecA, and,via a two-component system, FecIR (49), which is responsiveto FecA binding of its cognate siderophore (4, 49). Similarly,pseudobactin BN7/8 stimulates expression of its receptor, PupB,via FecIR homologues, dubbed PupIR (17). FecI is an extracytoplasmic-function(ECF) sigma factor (1) whose activity is controlled by FecR(30). Recently, a third example of this type of regulatory systemwas reported in Bordetella bronchiseptica, where bupIR geneproducts were shown to control expression of a putative siderophorereceptor, BfrZ (37). In an effort to understand the basis ofpyoverdine control of FpvA production in P. aeruginosa, then,FecIR homologues were sought in the pvd locus of the availablePAO genome (http://www.pseudomonas.com) (47).

MATERIALS AND METHODS

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Materials and Methods

Bacterial strains and growth media. Bacterial strains and plasmids used in this study are listedin Table 1. Routine growth for both P. aeruginosa and E. coliwas performed in Luria-Bertani (LB) medium (Luria broth base;Difco). Growth under iron-limited conditions was performed byusing iron-free BM2 succinate or glucose medium (35), whichwas made to be iron sufficient, as necessary, through the additionof 100 µM FeSO₄. Antibiotic selections used for P. aeruginosaincluded tetracycline (70 µg/ml in LB and 30 µg/mlin BM2 succinate), chloramphenicol (200 µg/ml in LB and30 µg/ml in BM2 succinate) and kanamycin (for ${Delta}$ aphA strainsonly, 100 µg/ml in LB). For E. coli, tetracycline wasused at 10 µg/ml (in LB) or 5 µg/ml (in BM2 glucose),chloramphenicol was used at 50 µg/ml (in LB) or 20 µg/ml(in BM2 glucose), and kanamycin was used at 50 µg/ml.

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TABLE 1. Bacterial strains and plasmids used in this study

DNA techniques. Basic DNA procedures, including restriction endonuclease digestions,ligations, transformations, and agarose gel electrophoresis,were performed as described previously (24). Plasmid DNA isolationwas performed by using the alkaline lysis method (24) or byusing a plasmid Midi kit (Qiagen, Mississauga, Ontario, Canada).Genomic DNA was extracted from P. aeruginosa by using the methodof Barcak et al. (3). DNA fragments for use in cloning wereextracted from agarose gels by using Prep-A-Gene (Bio-Rad Labs,Richmond, Calif.) in accordance with the manufacturer's instructions.Nucleotide sequencing of plasmid-borne DNA was carried out byCortec DNA Services, Inc. (Kingston, Ontario, Canada). Nucleotidesequence alignments were performed using the GENESTREAM website(http://xylian.igh.cnrs.fr/bin/align-guess.cgi) (33).

Cloning of fpvI and construction of a ${Delta}$ fpvI mutant. The fpvI gene was amplified by PCR by utilizing primers pff(5'-CATGGAATTCTGGTAGTTGGAAGGAATCCAGC-3'; the EcoRI site is underlined)and pfr (5'-AGCTGAATTCCAGTTGCCTGAGTCAATTCCAG-3'; the EcoRI siteis underlined). The PCR mixture contained 50 ng of P. aeruginosachromosomal DNA, 30 pmol of each primer, 0.2 mM (each) deoxynucleosidetriphosphate, 1 mM MgSO₄, and 3% (vol/vol) dimethyl sulfoxidein 1x ThermoPol buffer (New England Biolabs, Mississauga, Ontario,Canada), which was heated for 3 min at 95°C before the additionof 2 U of VentR DNA polymerase (New England Biolabs). The reactionwas processed for 30 cycles of 1 min at 95°C, 20 s at 58°C,and 1 min at 72°C, followed by 10 min at 72°C. The resultingamplicon was purified by a Qiaquick PCR purification kit (Qiagen),digested with EcoRI, and cloned into EcoRI-digested pRK415,to yield pAR001. Nucleotide sequencing confirmed that the fpvIgene was cloned in the same orientation as the lac promoter.

To construct ${Delta}$ fpvI mutants of P. aeruginosa, an internal deletionof the fpvI gene was first constructed in the gene replacementvector pK18mobsacB. This was accomplished by amplifying PCRproducts (by using the conditions and parameters described above)corresponding to sequence upstream and downstream of the deletionend points in fpvI by using primer pairs pflaeco (5'-GATCGAATTCATGCTGCCTCTCGCGATGTC-3';the EcoRI site is underlined) and pflakpn (5'-CGTAGGTACCGGCACTGAGGAATCGCAG-CA-3';the KpnI site is underlined) and pfrakpn (5'-CGTAGGTACCAACGCGATGAAGCACTGTC-3';the KpnI site is underlined) and pfrahind (5'-GACTAAGCTTGTGTTCCAGGTACTGGCTCTG-3';the HindIII site is underlined), respectively. The downstreamfragment was purified by using a Qiaquick PCR purification kit(Qiagen), digested with KpnI and HindIII, and cloned into KpnI-HindIII-restrictedpEX18Tc. The resulting vector was then digested with EcoRI andKpnI, and the EcoRI-KpnI-digested upstream fragment was clonedinto this vector, yielding the ${Delta}$ fpvI plasmid pAR002. The ${Delta}$ fpvIgene was excised from pAR002 by digestion with EcoRI and XmnI,and the resultant 1.8-kb fragment was cloned into EcoRI-XmnI-restrictedpK18mobsacB to yield pAR003. This vector was then transformedinto E. coli S17-1 and mobilized into P. aeruginosa strainsK1120 and K1203 by using a previously described protocol (45).By using chloramphenicol (5 µg/ml) as a counterselectiveagent, kanamycin-resistant transconjugants were recovered andsubsequently streaked onto LB agar containing 10% (wt/vol) sucrose.Sucrose-resistant colonies arising after 24 h of growth at 37°Cwere screened for the presence of a ${Delta}$ fpvI chromosomal deletionby colony PCR (42) with primers pff and pfr by using the conditionsdescribed above.

Whole-cell extracts. Whole-cell protein extracts were prepared from cultures of P.aeruginosa grown overnight in iron-sufficient or iron-deficientBM2 succinate medium following subculture (1:99 dilution) intothe same medium and growth to an optical density at 600 nm (OD₆₀₀)of approximately 0.7. Two 1.5-ml aliquots were centrifuged sequentiallyin the same microcentrifuge tube at 13,000 rpm (Biofuge pico;Heraeus Instruments) and resuspended in 400 µl of phosphate-bufferedsaline (1.7 mM NaH₂PO₄, 8.1 mM Na₂HPO₄, 145 mM NaCl). An equalvolume of loading buffer (4% [wt/vol] sodium dodecyl sulfate[SDS], 20% [vol/vol] glycerol, 250 mM Tris-HCl [pH 6.8]) wasthen added, after which the samples were boiled for 5 min andsonicated on ice for 10 s (by using setting 40 on a Vibra Cellsonicator [Sonic and Materials Inc., Danbury, Conn.]). Totalcell protein concentrations were determined using a BCA proteinassay kit (Pierce, Rockford, Ill.) according to the manufacturer'sinstructions.

SDS-polyacrylamide gel electrophoresis and immunoblotting. Whole-cell extract samples were electrophoresed on 10% (wt/vol)SDS-polyacrylamide gels (53) and electrotransferred onto Immobilon-Ppolyvinylidene difluoride membranes (Millipore) as describedpreviously (54). Equal loading of protein in all wells was confirmedby rapid Coomassie staining of duplicate gels (9). Membraneswere probed with a polyclonal anti-FpvA antibody as describedpreviously (36).

ß-Galactosidase assay. P. aeruginosa strains containing promoter-lacZ fusions weregrown in iron-limited BM2 succinate medium supplemented withthe appropriate antibiotics for approximately 18 h at 37°C.In some experiments, pyoverdine was included at 100 µg/ml.Cultures were then diluted 1:49 into the same medium withoutantibiotics and grown at 37°C to an OD₆₀₀ of $~$ 0.7 beforebeing assayed for ß-galactosidase activity as describedpreviously (25). To assess the impact of the cloned fpvI geneon fpvA-lacZ expression in a heterologous host, E. coli DH5 ${alpha}$ harboring pEJB3 and either pAR001 (which carries fpvI) or pAR005(vector control) was grown in iron-limited BM2 glucose minimalmedium with the appropriate antibiotics to an OD₆₀₀ of $~$ 0.5 beforebeing assayed for ß-galactosidase activity.

RNA isolation and RT-PCR. RNA was isolated by using the RNeasy RNA isolation kit (Qiagen)in accordance with the manufacturer's instructions. Sampleswere then treated with RQ1 DNase (Promega, Madison, Wis.) inaccordance with the manufacturer's instructions, and RNA wasquantitated by spectrophotometry. Reverse transcription (RT)-PCRwas carried out by using intragenic primers specific for fpvI(namely, piff [5'-ACTGGAATTCCAGCGAGCAGGAGTCGTCTT-3'] and pifr[5'-ACTGGAATTCTTGCGCAACAGGAAGGAAC-3']) and rpsL (namely, rpsL1[5'-GCAACTATCAACCAGCTG-3'] and rpsL2 [5'-GCTGTGCTCTTGCAGGTTGTG-3'])and the One Step RT-PCR kit (Qiagen) by following the manufacturer'sinstructions. PCR conditions were as described above, exceptthat the annealing temperature was 59°C. Non-RT controlswere run to ensure that no DNA contaminated the RNA samples.

RESULTS AND DISCUSSION

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Results and Discussion

Role of FpvI in fpvA expression. Examination of the Pseudomonas genome (Pseudomonas genome projectwebsite, http://www.pseudomonas.com) by using BLASTX (NationalCenter for Biotechnology Information webpage, http://www.ncbi.nlm.nih.gov/BLAST)revealed two open reading frames, PA2387 and PA2388, whose deducedproducts exhibited homology to FecIR and PupIR, and they weresubsequently designated fpvI and fpvR (in accordance with thenomenclature suggested by I. Lamont, University of Otago). ThefpvI and fpvR genes were transcribed divergently from one anotherand were localized within the so-called pvd locus between thepyoverdine biosynthetic genes pvdK (19) and pvdA (50). FpvIshowed almost 30.6% identity (59% overall similarity) to FecI(accession no. AAA23766), 31.6% identity (63.3% overall similarity)to BupI (accession no. CAB71123), and 29.5% identity (60.1%overall similarity) to PupI (accession no. CAA54870). To assessits role in FpvA production, then, an in-frame fpvI deletionwas constructed in P. aeruginosa strain K1120, yielding K2100.Although FpvA production increased substantially in FpvI⁺ strainsunder iron restriction (compare lanes 1 and 2 in Fig. 1A), lossof fpvI in K2100 abolished this iron-limited increase in FpvA(Fig. 1A, lane 3). FpvA production in iron-limited K2100 was,however, restored by the vector-borne fpvI gene present on plasmidpAR001 (Fig. 1A, lane 4), confirming a role for this probableECF sigma factor in FpvA expression under iron-limiting conditions.To assess whether FpvI was influencing FpvA at the level ofgene expression, an fpvA-lacZ fusion vector, pEJB3, was introducedinto FpvI⁺ and FpvI^- P. aeruginosa, and ß-galactosidaseactivity was determined for cells cultured under iron-limitingconditions. As seen in Table 2, loss of fpvI in K2100 reducesfpvA expression approximately fivefold relative to that of theFpvI⁺ parental strain, K1120. Moreover, the cloned fpvI generestored fpvA expression to wild-type levels in K2100 and stimulatedthe expression of the fpvA-lacZ fusion in E. coli (Table 2).In contrast, pvdD-lacZ expression, previously demonstrated tobe dependent on the PvdS ECF sigma factor (21), was not stimulatedby FpvI in E. coli (data not shown). These data are consistentwith FpvI directly and positively promoting fpvA gene expression.

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FIG. 1. Immunoblots of iron-limited P. aeruginosa whole-cell extracts developed with anti-FpvA antibodies. (A) Lane 2, K1120 (FpvI⁺); lane 3, K2100 (FpvI^-); lane 4, K2100 carrying pAR001 (FpvI⁺). An immunoblot of K1120 grown in iron-supplemented minimal medium is shown in lane 1. (B) Lane 1, K1120 (FpvI⁺); lanes 2 and 3, K1203 (FpvI⁺ PvdD^-); lanes 4 and 5, K2102 (FpvI^- PvdD^-); lane 6, K2102 carrying pAR001 (FpvI⁺ PvdD^-). Samples in lanes 3, 5, and 6 were prepared from cells supplemented with pyoverdine (100 µg/ml) during growth.

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TABLE 2. Influence of FpvI on fpvA-lacZ expression^a

Iron regulation of fpvI. Since many iron uptake and regulatory genes, including thatencoding the aforementioned PvdS ECF sigma factor, are themselvesregulated by iron, with regulation mediated by the Fur repressorprotein, it was of interest to assess regulation of fpvI byiron. By RT-PCR, expression of fpvI was clearly shown to increaseunder conditions of iron limitation (Fig. 2). Moreover, examinationof the nucleotide sequence upstream of fpvI revealed the presenceof a putative Fur box (Fig. 3, underlined), with 12 of 19 nucleotidesmatching the consensus Fur box sequence; indeed, Fur bindingto this site has been previously confirmed (fpvI was previouslyidentified as a Fur-regulated gene dubbed pig32 [31]). Thus,the observation that expression of fpvA is governed by an iron-regulatedprobable sigma factor explains the known iron regulation ofthis receptor gene despite the absence of a Fur box upstreamof the fpvA gene. This indirect iron regulation of gene expressionis reminiscent of the iron-regulated expression of the pyoverdinebiosynthetic (pvd) genes, which also lack Fur boxes and whoseexpression is governed by the iron-regulated ECF sigma factorPvdS (20, 21, 32, 52).

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FIG. 2. fpvI expression in P. aeruginosa strain PAO1 K767, measured by RT-PCR of RNA (4 ng) isolated from cells cultivated under iron-sufficient (odd-numbered lanes) and iron-deficient (even-numbered lanes) conditions. The reactions were carried out for 26 (lanes 1 and 2), 27 (lanes 3 and 4), 28 (lanes 5 and 6), 29 (lanes 7 and 8), and 30 (lanes 9 and 10) cycles. fpvI and the rpsL (control) products are highlighted. Lane 11, 100-bp ladder with a 500-bp marker highlighted.

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FIG. 3. Nucleotide sequence of the 5' upstream region of fpvI, highlighting a putative Fur box. The Fur box (underlined) and FpvI translational initiation codon (italicized) are highlighted. Matches to the consensus Fur box sequence are capitalized.

Involvement of pyoverdine in FpvI-mediated fpvA expression. In order to ascertain whether FpvI mediates the positive influenceof pyoverdine on FpvA production, an fpvI deletion was createdin a pyoverdine-deficient derivative of P. aeruginosa, K1203,and the ability of exogenously added pyoverdine to promote FpvAproduction in the resulting strain, K2102, was assessed. Immunoblottingwith an anti-FpvA antibody confirmed previous findings thatFpvA production was reduced in a pyoverdine-deficient (but FpvI⁺)strain (Fig. 1B, lane 2; compare with lane 1) and can be restoredby the addition of exogenous pyoverdine (Fig. 1B, lane 3). Incontrast, pyoverdine did not restore FpvA production in theFpvI^- strain K2102 (Fig. 1B, lane 5; compare with lane 4). Introductionof the cloned fpvI gene on plasmid pAR001 did, however, restorepyoverdine stimulation of FpvA production in this mutant (Fig.1B, lane 6). Again, this effect occurred at the level of fpvAgene expression, with pyoverdine enhancing expression of thefpvA-lacZ fusion in the FpvI⁺ strain, K1203, but not in theFpvI^- strain, K2102 (Table 2). The observation that FpvI mediatesthe positive influence of pyoverdine on fpvA gene expressionis reminiscent of PvdS and its mediation of the positive influenceof this siderophore on pvd gene expression, the latter in aprocess involving the receptor itself and an anti-sigma factor,FpvR (18, 41). Pyoverdine-dependent FpvI-mediated stimulationof fpvA expression appears also to be controlled by FpvA andFpvR (I. L. Lamont, personal communication).

ACKNOWLEDGMENTS

The ${Delta}$ pvd strain K1203 was engineered by using a ${Delta}$ pvdD plasmidprovided by I. L. Lamont (University of Otago).

This work was supported by an operating grant from the CanadianInstitutes of Health Research (formerly the Medical ResearchCouncil of Canada) to K.P. K.P. is a Canadian Cystic FibrosisFoundation (CCFF) Scholar. G.A.R. is the recipient of a CCFFgraduate student scholarship.

FOOTNOTES

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Queen's University, Botterell Hall, Room 813, Stuart St., Kingston, Ontario K7L 3N6, Canada. Phone: (613) 533-6677. Fax: (613) 533-6796. E-mail: poolek{at}post.queensu.ca.

REFERENCES

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