Different Responses of Pyoverdine Genes to Autoinduction in Pseudomonas aeruginosa and the Group Pseudomonas fluorescens-Pseudomonas putida

We investigated the regulation of the psbA and pvdA pyoverdinebiosynthesis genes, which encode the L-ornithine N⁵-oxygenasehomologues in Pseudomonas strain B10 and Pseudomonas aeruginosaPAO1, respectively. We demonstrate that pyoverdine_B10, as theend product of its biosynthetic pathway, is a key participantof the control circuit regulating its own production in Pseudomonasstrain B10. In P. aeruginosa PAO1, however, pyoverdine_PAO1 hasno apparent role in the positive regulation of the pvdA gene.

INTRODUCTION

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Introduction

The competition for iron is a main determinant for the dynamicsof microbial populations in natural ecosystems. Aerobic bacterialiving in neutral environments are normally faced with the nutritionaliron deficit resulting from the low solubility of iron in itsoxidized state (13). Likewise, bacterial parasites, which multiplywithin higher organisms, must counteract the iron deficiencyimposed by the activity of the host's iron binding proteins(15). Iron withholding also affects the interactions betweenmicrobial populations in the rhizosphere (14).

Pyoverdines (also referred to as pseudobactins), the fluorescentsiderophores produced by the rRNA group I species of genus Pseudomonas,constitute a large family of iron chelators that differ in termsof the length and composition of a hydroxamate-containing oligopeptidejoined to a structurally conserved dihydroxyquinoline chromophore(1). Due to their high affinity for ferric iron, pyoverdinescan suppress the growth of deleterious rhizomicroorganisms,which lack uptake specificities for ferripyoverdine complexes,thereby contributing to the plant defense from parasitic infection(2, 14).

In Pseudomonas spp. and many other bacteria, regulation of irontransport is primarily controlled by the Fur repressor protein(22). When the level of intracellular iron is high, transcriptionof iron-repressible genes is blocked through the binding ofFur-Fe(II) complexes to a highly conserved sequence (the Furbox) located within the promoters of these genes (5). However,Fur is not the only regulator of iron uptake in fluorescentpseudomonads (24). In Pseudomonas aeruginosa, the rRNA groupI type species, expression of pyoverdine_PAO1 (pvd) genes iscontrolled by iron through a cascade of negative and positiveregulatory proteins (Fig. 1). Briefly, Fur acts as a repressorof the pvdS gene, encoding an extra cytoplasmic function (ECF)sigma factor, called PvdS, which is required for transcriptioninitiation at the promoters of pvd genes (4, 9). A DNA sequenceelement, the iron starvation box (ISB), has been identifiedin PvdS-dependent promoters and has been shown to participatein sequence-specific promoter recognition by PvdS, acting asa -35-like sequence element (27). Several Fur-controlled alternativesigma factors, structurally related to PvdS, have been identifiedin fluorescent Pseudomonas spp. and have been found to be involvedin pyoverdine synthesis and/or uptake (7, 20, 23). An additionallevel of complexity of the siderophore regulatory pathway inthese species is inferred by the positive effect of ferric siderophorecomplexes on the expression of cognate biosynthesis genes. Siderophore-regulatedresponses have previously been demonstrated for the pyochelin-salicylateoperons of P. aeruginosa and for the pyoverdine gene systemsof Pseudomonas putida WCS358 and Pseudomonas fluorescens M114(3, 16, 23, 24).

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FIG. 1. Regulatory models of pyoverdine genes in P. aeruginosa PAO1 and in Pseudomonas strain B10. (A) In iron-replete P. aeruginosa PAO1 cells, transcription of pvdS is prevented by the binding of dimeric Fur-Fe(II) complexes to Fur boxes overlapping the pvdS promoter. In the absence of sufficient iron, release of Fur from DNA allows pvdS to be transcribed. The ECF sigma factor PvdS mediates recognition of the ISB, thereby conferring specificity to core RNA polymerase (cRNAP) for transcription initiation at the pvd promoters. (B) In Pseudomonas strain B10, expression of the psbA gene is similarly regulated by the Fur-controlled ECF sigma factor PsbS. An additional level of regulation is provided by exogenous pyoverdine_B10, which exerts a positive induction effect on expression of the psbA biosynthetic gene.

The pyoverdine_B10 biosynthesis gene psbA, encoding the enzymeL-ornithine (L-Orn) N⁵-oxygenase (PsbA) in the plant growth-promotingPseudomonas strain B10, has previously been characterized inour laboratory (2). PsbA expression is regulated at the transcriptionallevel by a Fur-controlled ECF sigma factor, designated PsbS(8). Herein, we provide evidence that pyoverdine_B10 itself playsa crucial role in the expression of the psbA gene. The comparisonof autogenous regulation of pyoverdine biosynthesis in Pseudomonasstrain B10 and P. aeruginosa PAO1 highlighted relevant differencesbetween the two species.

The bacterial strains and plasmids used in this study are listedin Table 1. The media and growth conditions have been describedpreviously (6, 9, 25).

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TABLE 1. Bacterial strains and plasmids used in this study

Characterization of the psbA promoter in Pseudomonas strain B10.

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Characterization of the psba...

To define the structure of the psbA promoter (PpsbA), the initiationsites of the psbA transcript were determined by primer extensionanalysis. The primer PEpsbARV (5'-GGGCGATGCAATTGCCGTTGTCAT-3')was end labeled with [ ${gamma}$ -³²P]ATP and used for reverse transcriptionof total RNA from iron-deprived Pseudomonas strain B10 cellsas described elsewhere (9). The unlabeled primer was used tosequence the DNA region upstream from the psbA gene from plasmidpCA ${Delta}$ Sh with a T7 sequencing kit (Pharmacia) and [ ${alpha}$ -³²P]dATP. The5' ends of the psbA mRNA were mapped at four distinct sites(Fig. 2). The most abundant transcripts, T3 and T4, originateat positions -44 and -48 relative to the psbA start codon, respectively,while two minor transcripts, T1 and T2, originate at positions-87 and -89, respectively. No primer extension product was detectedwith total RNA from bacteria grown in iron-rich medium (datanot shown), consistent with the observation that psbA is nottranscribed in iron-rich cells (2). The initiation pattern ofpsbA mRNA is consistent with the presence of multiple transcriptionstart sites in the homologous pvdA promoter (PpvdA) and in P.aeruginosa iron-regulated promoters directly or indirectly controlledby the PvdS sigma factor, including pvdD, regAB, and toxA (9,17, 22). As shown in Fig. 2, the 158-bp DNA sequence regionpreceding the psbA start codon is 68% identical to the correspondingPpvdA region (9). Both sequences contain regulatory motifs sharedby ECF-dependent promoters, namely the ISB and the TCCTA element,which is also present in the corresponding region of algD andalgU promoters of P. aeruginosa (18). It was also noticed thatthe psbA T2 and T4 transcripts correspond to the pvdA T1 andT2 transcripts, respectively. Interestingly, in Pseudomonasstrain B10, the most abundant psbA transcript was T4, while,under the same experimental conditions, the major pvdA transcriptwas T1 (9). The alignment highlights the presence of a 14-nucleotide(nt) deletion and an 18-nt insertion at positions -137 and -49upstream of the psbA start codon, respectively. Due to the presenceof the 18-nt insertion in PpsbA, the T4 start point is locatedat a longer distance from the ISB compared with the T2 startpoint of pvdA (Fig. 2). This insertion is endowed with remarkablefeatures, consisting of two heptameric direct repeats (TCAGGCC),followed by a third partially conserved repeat (cCAGGCt).

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FIG. 2. Structural analysis of the psbA promoter. (A) Alignment of the 158-nt region preceding the Pseudomonas strain B10 psbA start codon with the P. aeruginosa pvdA promoter. Identical nucleotides are capitalized and in boldface. The bent arrows indicate the minor (T1 and T2) and major (T3 and T4) transcription start sites of the psbA gene and the direction of transcription. The ISB and the TCCTA conserved motifs, which overlap the -35 and -10 regions, respectively, are underlined. The potential ribosome binding sites (RBS) and the transcriptional start codons (f-Met) of psbA and pvdA are underlined with dots. The locations of the conserved (solid arrows) and partially conserved (dotted arrow) direct repeats are indicated below the psbA sequence. Arrowheads below the pvdA sequence mark the positions of the 5' ends of the pvdA T1 and T2 transcripts (9). (B) Localization of the transcriptional start sites of psbA. The asterisk indicates primer extension analysis of the psbA mRNA carried out with the 5'-end-labeled oligonucleotide PEpsbARV. Lanes T, C, G, and A represent sequencing ladders of pCA ${Delta}$ Sh with the same oligonucleotide. These reactions were run in parallel with primer extension products to determine exactly the 5' ends of the transcripts. The arrows on the left indicate the origins of psbA transcripts T1, T2, T3, and T4.

Activity of psbA and pvdA promoters in Pseudomonas strain B10 and P. aeruginosa.

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Activity of psba and...

The structural similarity between the PpsbA and PpvdA, combinedwith previous evidence that PsbS and PvdS are functionally interchangeable(8), led us to investigate promoter activity in Pseudomonasstrain B10 and P. aeruginosa PAO1. For this purpose, the ß-galactosidaselevels expressed by pMP220::PpsbA and pMP220::PpvdA fusionswere measured in P. aeruginosa PAO1 and Pseudomonas strain B10cells grown under low- and high-iron conditions. Both promotersshowed similar activities in iron-poor cultures of P. aeruginosaPAO1 (Table 2). Comparable levels of ß-galactosidase(ca. 10,000 U) were also expressed by iron-limited culturesof Pseudomonas strain B10 carrying the homologous promoter fusion(pMP220::PpsbA), while a dramatic (>90%) reduction in reportergene activity was observed for the heterologous pMP220::PpvdAfusion (Table 2). These results suggest that the transcriptionalapparatus of Pseudomonas strain B10 discriminates between thehomologous promoter (PpsbA) and the heterologous promoter (PpvdA),the latter being less efficiently recognized.

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TABLE 2. Activity of PpsbA::lacZ and PpvdA::lacZ transcriptional fusions in wild-type Pseudomonas strain B10 and P. aeruginosa PAO1 and in isogenic pyoverdine-defective mutants

Pyoverdine-dependent regulation of pvdA and psbA promoters.

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Pyoverdine-dependent regulation...

Preliminary reports from independent laboratories have relatedthe down-regulation of pyoverdine promoters in pyoverdine biosyntheticmutants to pyoverdine-mediated induction of cognate siderophoregenes (3, 23). On this basis, we compared the pyoverdine-regulatedresponses in wild-type Pseudomonas strain B10 and P. aeruginosaPAO1 and in their isogenic pyoverdine-defective derivatives,B10CA1 (psbA::cat) and PALS124 (pvdA) (2, 26). Under low-ironconditions, the activities of both PpsbA::lacZ and PpvdA::lacZfusions were strongly reduced in Pseudomonas strain B10CA1 comparedwith those in the parental strain, and this effect was reversedby addition of 50 µM pyoverdine_B10 to the culture medium(Table 2). In contrast, the activities of both promoter fusionswere similar in P. aeruginosa PAO1 and in the PALS124 mutant.The pyoverdine-insensitive behavior of the PpvdA promoter wasconfirmed in P. aeruginosa mutants blocked at later steps ofpyoverdine biogenesis, including PALS106 (pvdC1) and PALS125(pvdC3) (Table 2). These results were supported by Western blotanalysis of PvdA expression in the pvd mutants (Fig. 3). Underconditions of iron deficiency, comparable levels of PvdA weresynthesized by P. aeruginosa PAO1, PALS106, and PALS125, irrespectiveof the presence of exogenous pyoverdine_PAO1 in the culture medium(Fig. 3). As expected, addition of 100 µM FeCl₃ to theiron-poor medium completely repressed PpvdA::lacZ activity (Table2) and PvdA expression (Fig. 3). Therefore, it can be deducedthat homologous pyoverdine is involved in the activation ofthe psbA promoter in the Pseudomonas strain B10 system, whileit plays no apparent role in positive regulation of the pvdAbiosynthesis gene in P. aeruginosa PAO1. This effect cannotbe ascribed to pyoverdine_B10-mediated induction of the psbSgene, since both pvdS::lacZ and psbS::lacZ promoter fusions(as in plasmids pMP220::PpvdS and pMP220::PpsbS) were foundto be equally expressed and iron regulated in P. aeruginosaPAO1 and Pseudomonas strain B10 wild-type strains and in theirL-Orn N⁵-oxygenase-defective mutants (data not shown).

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FIG. 3. Western blot analysis of iron-regulated PvdA expression in wild-type and pvd mutants of P. aeruginosa PAO1. Immunodetection of the 50-kDa PvdA protein in crude lysates ( ${cong}$ 20 µg of proteins per lane) of exponentially grown bacteria (A₆₀₀ ${cong}$ 0.5) probed with an anti-PvdA mouse antiserum was performed as previously described (2). Cultures were performed in KB medium supplemented or not with either 100 µM FeCl₃ [Fe(III)] or 50 µM pyoverdine_PAO1 (pvd_PAO1), as indicated below each lane. Lanes: 1 to 3, P. aeruginosa PAO1 (wild type); 4 to 6, PALS106 (pvdC1); 7 to 9, PALS125 (pvdC3). The position of PvdA is indicated on the left.

Conclusions.

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Conclusions.

The data just presented demonstrate that pyoverdine_B10, as theend product of its biosynthesis pathway, is a key participantin the control circuit regulating its own production in Pseudomonasstrain B10. In P. aeruginosa PAO1, however, extracellular pyoverdine_PAO1has no apparent role in the positive regulation of the homologouspvdA gene see Fig. 1 for a comparison between the two systems).The lack of pvdA response to exogenous pyoverdine is consistentwith the observation that pvd mutants blocked in late stepsof pyoverdine_PAO1 biogenesis produce wild-type levels of hydroxamatenitrogen in iron-poor medium (26). This implies that PvdA isequally expressed in the pyoverdine_PAO1-proficient and -deficientbackgrounds, and immunoblot analysis of PvdA expression in thewild-type and pvd mutants corroborates this conclusion.

Despite the differences in individual activity between PpsbAand PpvdA, which are likely to reflect their structural diversity,both promoters were found to respond positively to homologouspyoverdine in the Pseudomonas strain B10 system, but not inP. aeruginosa. The mechanism by which pyoverdine_B10 increasespsbA expression does not involve up-regulation of psbS transcription,implying that sensing of pyoverdine_B10 could result in posttranscriptionalactivation of the PsbS sigma factor. Siderophore-dependent inductionhas been elucidated in the pupIR-pupB system of P. putida WCS358.The Fur-controlled pupIR operon encodes a surface-signalingsystem. PupI is an ECF sigma factor required for the expressionof pupB, encoding the receptor for heterologous pyoverdine_BN8.PupR up-regulates pupB expression through activation of PupIin the presence of ferripyoverdine_BN8, but it prevents the PupI-dependenttranscription of pupB in its absence (7). Sequencing of thecomplete genomes of P. fluorescens and P. putida (availableat http://www.jgi.doe.gov/and http://www.ncbi.nlm.nih.gov/,respectively) disclosed the presence in these species of multiplegenes encoding putative PupR-like proteins. Whether a similarregulatory device also exists in Pseudomonas strain B10, itwould account for modulation of PsbS activity through signalingof ferripyoverdine_B10 binding to its outer membrane receptor(10). This would ensure up-regulation of psbA expression underconditions in which pyoverdine_B10 is effective in deliveringiron to the cell: namely, when ferripyoverdine_B10 is engagedwith its receptor.

ACKNOWLEDGMENTS

This investigation was supported by grants from the Departmentof Biology, University of Roma Tre, and from the Italian Ministryof Health (targeted projects 1999 and 2000) to P.V.

FOOTNOTES

* Corresponding author. Mailing address: Dipartimento di Biologia, Università di Roma Tre, Viale G. Marconi 446, 00146 Rome, Italy. Phone: 39.6.5517.6347. Fax: 39.6.5517.6321. E-mail: visca{at}bio.uniroma3.it.

REFERENCES

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