Molecular basis of pyoverdine recycling in aeruginosa

Francesco Imperia,b, Federica Tiburzia,b, and Paolo Viscaa,b,1

aDepartment of Biology, University ‘‘Roma Tre,’’ Viale G. Marconi 446, 00146 Roma, Italy; and bMolecular Microbiology Unit, National Institute for Infectious Disease Istituto Di Ricovero e Cura a Carattere Scientifico ‘‘Lazzaro Spallanzani’’, Via Portuense 292, 00149 Roma, Italy

Edited by E. Peter Greenberg, University of Washington School of Medicine, Seattle, WA, and approved September 25, 2009 (received for review August 3, 2009) The siderophore pyoverdine (PVD) is a primary of (IM) transporter PvdE, which is essential for PVD production the human pathogenic bacterium , acting (9). In addition, the pvd locus contains an for a tripartite as both an carrier and a virulence-related signal molecule. By system of the ABC superfamily (PA2389–90-OpmQ), exploring a number of P. aeruginosa candidate systems for PVD whose expression mirrors that of PVD synthesis enzymes, being , we identified a tripartite ATP-binding cassette efflux controlled by the PVD-specific sigma factor PvdS. Deletion of transporter, here named PvdRT-OpmQ, which translocates PVD this system reduces, but does not abrogate, PVD production (4). from the periplasmic space to the extracellular milieu. We show PVD-mediated iron uptake occurs by binding of the ferri-PVD this system to be responsible for recycling of PVD upon internal- complex to the outer membrane (OM) FpvA (10, 11), ization by the cognate outer-membrane receptor FpvA, thus mak- while a secondary PVD receptor, called FpvB, provides a minor ing PVD virtually available for new cycles of iron uptake. Our data contribution to PVD uptake (12). Iron release from PVD has exclude the involvement of PvdRT-OpmQ in secretion of de novo been shown to occur in the , and that apo-PVD is then synthesized PVD, indicating alternative pathways for PVD export recycled to the extracellular milieu (13, 14). However, no protein and recycling. The PvdRT-OpmQ transporter is one of the few candidates have been proposed for these processes and further secretion systems for which substrate recognition and extrusion studies are required to elucidate the fate of iron and PVD occur in the periplasm. Homologs of the PvdRT-OpmQ system are following dissociation in the periplasm (15). present in genomes of all fluorescent pseudomonads sequenced so Here, we demonstrate that the tripartite efflux system far, suggesting that PVD recycling represents a general energy- PA2389–90-OpmQ is implicated in PVD recycling by P. aerugi- saving strategy adopted by natural Pseudomonas populations. nosa, and that PVD recycling and export probably involve alternative pathways. ATP-binding-cassette transporter ͉ periplasm ͉ iron ͉ fluorescent Pseudomonas ͉ secretion Results PA2389, PA2390, and opmQ Mutants Accumulate PVD in the Periplasm. seudomonas aeruginosa is among the most dreaded Gram- In a preliminary search for candidate PVD secretion systems, we compared the PVD levels between culture supernatants of negative in the hospital setting and the main cause P wild-type P. aeruginosa PAO1 and transposon mutants in of lung decline and death in patients suffering from cystic fibrosis encoding the MexAB-OprM, PvdE, and PA2389–90-OpmQ (CF). In response to iron depletion, P. aeruginosa utilizes the transport systems [supporting information (SI) Table S1]. No endogenous siderophore pyoverdine (PVD) as the primary iron significant growth differences between wild-type PAO1 and source. In addition to its role in nutrition, PVD is implicated in putative PVD secretion mutants were observed under both control, -to-cell communication, and virulence reg- iron-deplete (iron-deplete Casamino acids or DCAA medium) ulation (1). By a mechanism known as surface signaling, PVD and -replete (DCAA medium plus 50 ␮M FeCl ) conditions (Fig. tunes the expression of virulence factors, including exotoxin A, 3 S1). In the iron-deplete medium, PVD production was: (i) exoprotease PrpL, and PVD itself (2). Accordingly, PVD is marginally affected by mutation in any of the mexA, mexB, and essential for P. aeruginosa virulence in several animal models of oprM genes; (ii) significantly reduced (approximately 50–60% of infection, and it has been found at biologically significant the wild-type levels) by mutation in any of the PA2389, PA2390, concentrations in the sputum from CF patients (reviewed in refs. and opmQ genes; and (iii) completely abolished in the pvdE and 1 and 3). pvdA mutants (Fig. 1A). PVD-related are produced by all fluorescent We took advantage of the fluorescent properties of apo-PVD pseudomonads, and consist of a conserved fluorescent chro- to determine its amount in cell lysates of wild-type PAO1 and mophore linked to a short differing among species and isogenic mutants by fluorimetric measurements (see Fig. 1A). strains. PVD synthesis and transport have been studied most The mexA, mexB, and oprM mutants showed intracellular levels intensively in the type strain P. aeruginosa PAO1. Almost all of PVD comparable to or even lower than those of the wild type, genes required for PVD synthesis and uptake cluster at a single consistent with their profile of PVD production. PVD was (pvd) locus in the PAO1 genome (4). PVDs are typically undetectable in cell lysates of the pvdE mutant, suggesting that produced by nonribosomal , through a pathway PvdE is not involved in PVD secretion or, alternatively, that it involving several synthetases and accessory enzymes that gen- directs the export of a nonfluorescent PVD precursor named erate nonproteinogenic precursors (1). Other ferribactin (1, 7). Conversely, PA2389, PA2390, and opmQ products are also essential for PVD production, but their actual role has not yet been established. Some of these have a periplas- mic localization (5, 6), in accordance with preliminary evidence Author contributions: F.I. and P.V. designed research; F.I. and F.T. performed research; F.I. of PVD maturation in the periplasmic compartment (7). and P.V. analyzed data; and F.I. and P.V. wrote the paper. The mechanism of PVD secretion is so far unknown. Involve- The authors declare no conflict of interest. ment of the multidrug efflux pump MexAB-OprM in PVD This article is a PNAS Direct Submission. secretion has been proposed (8), but not confirmed yet. One 1To whom correspondence should be addressed. E-mail: [email protected]. candidate for PVD transport from the to the This article contains supporting information online at www.pnas.org/cgi/content/full/ periplasm is the ATP-binding-cassette (ABC) inner membrane 0908760106/DCSupplemental.

20440–20445 ͉ PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908760106 Downloaded by guest on September 26, 2021 MICROBIOLOGY

Fig. 1. Extracellular and cell-associated PVD in candidate PVD export mutants of P. aeruginosa PAO1. Wild-type and mutant strains were grown for 14 h at 37 °C in DCAA, or in DCAA plus 50 ␮M FeCl3 (ϩFe). (A) Relative levels of extracellular PVD (black bars; Left y axis) were determined spectrophotometrically in culture supernatants. Intracellular PVD was determined fluorimetrically in 1-ml cell lysates from 5 ϫ 109 cells (white bars; Right y axis). Values are mean (Ϯ SD) of three independent assays. (B) Confocal microscopy of P. aeruginosa mutant cells. For each strain/condition, the left and right panels show the visible and the corresponding 405-nm laser line confocal microscopy pictures, respectively.

mutants accumulated five- to sixfold higher amounts of PVD mutants, intracellular PVD was detected in living cells by than wild-type PAO1, suggesting an involvement of these genes confocal laser-scanning microscopy (Fig. 1B). The in PVD export. Therefore, the PA2389 and PA2390 genes were emission levels of pvdR-, pvdT-, and opmQ-mutant cells largely renamed pvdR and pvdT, respectively, to emphasize their role in exceeded that of wild-type cells. No fluorescence was detected PVD transport or recycling. in the PVD-defective pvdA mutant or in iron-replete wild-type To visualize PVD accumulation by pvdR, pvdT, and opmQ cells (see Fig. 1B and Fig. S2), validating the identification of the

Imperi et al. PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 ͉ 20441 Downloaded by guest on September 26, 2021 Next, we sought to investigate the involvement of the PvdRT- OpmQ efflux system in recycling PVD after FpvA-mediated ferri-PVD uptake. To this aim, iron-deplete cells of pvdA, pvdApvdR, and fpvApvdA mutants were incubated with the nonfluorescent ferri-PVD complex (1 ␮M). Recycling of apo- PVD was monitored fluorimetrically as the appearance of PVD-specific fluorescence in culture supernatants (14). The pvdA strain efficiently recycled apo-PVD in the culture medium, while both the pvdApvdR and fpvApvdA double mutants did not (Fig. 3D). Nevertheless, pvdApvdR cells accumulated apo-PVD (see Fig. 3D), indicating that they are able to remove iron from ferri-PVD in the periplasm but are unable to recycle apo-PVD. Fig. 2. Subcellular localization of cell-associated PVD in pvdR, pvdT, and Moreover, pvdA and pvdApvdR cells showed similar ability to use opmQ mutants. Strains were grown for 14 h at 37 °C in DCAA. PVD was PVD as an iron source, as determined by a feeding assay in which determined fluorimetrically in 1-ml periplasmic (gray bars) and spheroplasts (white bars) fractions from 5 ϫ 109 cells. Values are mean (Ϯ SD) of three exogenously added PVD was used to sustain bacterial growth independent assays. under conditions of extreme iron restriction (DCAA plus 600 ␮M dipyridyl) (Fig. 3E). Taken together, our results substantiate the hypothesis of Greenwald et al. (14), according to which PVD cell-associated fluorescent molecule as apo-PVD. Once more, recycling and PVD-iron dissociation in the periplasm are inde- mexA-, mexB-, and oprM-mutant cells appeared similar to wild- pendent processes. type PAO1, thus excluding the involvement of MexAB-OprM in PVD export. No fluorescence was detected in pvdE-mutant cells. The PvdRT-OpmQ System Is Not Involved in Export of Endogenous To determine the cellular compartment where PVD is accu- PVD. To assess the involvement of the PvdRT-OpmQ efflux mulated by pvdR, pvdT, and opmQ mutants, cell-fractionation system in secretion of de novo synthesized PVD, we investigated experiments were performed. For this purpose, the periplasm the effect of PvdRT-OpmQ inactivation in an FpvA-deficient was extracted from P. aeruginosa cells and the amount of PVD mutant, which retains the ability to produce PVD (2) but is was compared between the periplasmic fraction and the resulting severely impaired in its uptake (see Fig. 3). Remarkably, the spheroplasts. Notably, nearly all intracellular PVD was associ- amount of PVD stored in the periplasm did not differ signifi- ated with the periplasmic fraction of pvdR, pvdT, and opmQ cantly between fpvA and fpvApvdR mutants, while it was much mutants (Fig. 2), irrespective of the mutation. Complementation lower than that of PAO1 and pvdR mutant (Fig. 4A). Extracel- of the pvdR, pvdT, and opmQ mutations with the plasmid-borne lular PVD levels were also comparable between fpvA and pvdRT-opmQ operon restored nearly wild-type levels of both fpvApvdR (see Fig. 4A), denoting no defect in secretion of de extracellular and periplasmic PVD, as demonstrated by fluori- novo produced PVD by the fpvApvdR mutant. Accordingly, metric and microscopic analyses (see Figs. 1 and 2). Therefore, confocal microscopy failed to reveal significant intracellular the entire PvdRT-OpmQ system (i.e., the IM ABC transporter accumulation of PVD by both fpvA and fpvApvdR mutants (Fig. PvdT, the periplasmic fusion protein PvdR, and the OM porin 4B). The barely detectable fluorescence shown by fpvApvdR cells OpmQ) appears to be involved in the transport of PVD from the was comparable to that of a fpvApvdRpvdA triple mutant fed with periplasm to the extracellular milieu. exogenous PVD (see Fig. 4B), suggesting that the weak fluo- rescence detectable in the fpvApvdR background originates from The PvdRT-OpmQ System Is Responsible for PVD Recycling. PVD- impaired recycling of some exogenous PVD acquired via an mediated iron uptake occurs through the OM receptor FpvA, FpvA-independent route, likely through the secondary receptor which translocates ferri-PVD into the periplasmic space. While FpvB. Indeed, no residual fluorescence was detected in a iron is internalized into the cytoplasm, PVD is recycled to the tonB1pvdR double mutant (see Fig. S2), in which receptor- extracellular milieu by a yet unknown mechanism (13, 14). dependent siderophore uptake is completely abrogated (16). Because the PvdRT-OpmQ efflux system is implicated in PVD In conclusion, these results indicate that periplasmic PVD export from the periplasmic space, its function in PVD recycling accumulation by PvdRT-OpmQ-deficient P. aeruginosa cells was investigated. To this aim, the pvdA mutant and the pvdApvdR originates from impaired recycling of exogenous PVD, rather and fpvApvdA double mutants, all defective in PVD synthesis, than from a defect in secretion of endogenous PVD. were grown in iron-deplete medium supplemented or not with 50-␮M exogenous PVD. Discussion Confocal microscopy and fluorimetric measurements of PVD Prerequisites for a system to be implicated in any step of PVD in subcellular fractions showed that the pvdApvdR double mu- secretion are the accumulation of intracellular PVD by the tant accumulated in the periplasm approximately sixfold higher corresponding mutants, together with reduced or impaired PVD amounts of PVD than the pvdA mutant (Fig. 3 A and B). secretion. We therefore addressed the role of MexAB-OprM, Periplasmic PVD levels were strongly reduced in the fpvApvdA PvdE, and PvdRT-OpmQ systems in PVD secretion by measur- double mutant lacking the FpvA receptor for the uptake of ing the amount of secreted PVD and the level of PVD-related extracellular PVD, and completely absent in the pvdApvdR fluorescence retained by P. aeruginosa cells. Our results rule out mutant incubated without PVD (see Fig. 3 A and B). The kinetics any significant contribution of the MexAB-OprM pump to PVD of exogenous PVD (50 ␮M) accumulation by iron-deplete cells secretion (see Fig. 1), while highlighting an essential role of PvdE of pvdA, pvdApvdR, and fpvApvdA mutants were consistent with in PVD biogenesis, as shown by the PVD-deficient phenotype of the above results. In pvdA cells, the amount of internalized PVD the pvdE mutant. In fact, PVD-related fluorescence was unde- reached a plateau between 30 and 60 min of incubation with tectable in pvdE mutant cells (see Fig. 1), and no hydroxamate- exogenous PVD, while pvdApvdR cells accumulated PVD for an containing PVD precursors could be detected in pvdE culture additional hour, attaining more than threefold higher concen- supernatants (SI Text). We therefore suggest that PvdE could tration of intracellular PVD than pvdA cells. In contrast, export the nonfluorescent PVD precursor ferribactin from the fpvApvdA cells accumulated very low levels of PVD (Fig. 3C), as cytosol to the periplasm without being involved in the secretion expected for a PVD uptake mutant. of mature PVD into the extracellular milieu. Accordingly,

20442 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908760106 Imperi et al. Downloaded by guest on September 26, 2021 MICROBIOLOGY

Fig. 3. PVD recycling from the periplasmic space. (A) Confocal microscopy of P. aeruginosa cells grown for 14 h at 37 °C in DCAA with or without 50-␮M exogenous PVD. For each strain/condition, the left and right panels show the visible and the corresponding 405-nm laser line confocal microscopy pictures, respectively. (B) Subcellular localization of PVD recycling. PVD was determined in periplasmic (gray bars) and spheroplasts (white bars) fractions as described in Fig. 2. (C) Kinetics of intracellular accumulation of PVD by iron-deplete cells incubated with 50-␮M exogenous PVD. (D) PVD recycling (white symbols) and PVD accumulation (black symbols) by iron-deplete intact cells incubated with 1 ␮M ferri-PVD. Symbols for (C) and (D) are: pvdA, diamonds; pvdApvdR, squares; fpvApvdA, circles. (E) Utilization of PVD as iron source by pvdA (black bars) and pvdApvdR (white bars) measured as growth yields after 14 h in DCAA plus 600 ␮M dipyridyl and exogenously-added PVD (0–80 ␮M). All values are mean (Ϯ SD) of three independent assays.

maturation of the PVD chromophore has been proposed to indicating that the PvdRT-OpmQ system is involved in secretion occur in the periplasm (7). of PVD from the periplasmic space to the extracellular milieu. Only mutation of the PvdRT-OpmQ exporter resulted in both Previous studies localized the substrate binding site of some reduction of extracellular PVD levels and remarkable intracel- resistance-nodulation division-type (RND) efflux pumps in the lular accumulation of PVD (see Fig. 1). PvdRT-OpmQ displays periplasmic compartment, and suggested that substrate extru- the typical architecture of Gram-negative tripartite ABC-type sion from the periplasm could represent a strategy to protect the exporters, consisting of an IM component with both ATPase and cytoplasm from potential hazards (17–19). This work provides permease domains, a periplasmic membrane fusion protein, and substantial evidence that ABC exporters can also extrude sub- a TolC-like OM protein (see SI Text for details). The pvdRT- strates from the periplasm, in line with a previous report showing opmQ gene cluster was found to be iron-regulated and under the that the macrolide efflux pump MacAB-TolC of Escherichia coli tight control of the PVD-specific PvdS sigma factor (Table S2), is also implicated in secretion of periplasmic enterotoxin II in line with previous transcriptomics data (4). (20). Although PvdRT-OpmQ and MacAB-TolC share remark- Cell fractionation assays showed that PVD is accumulated in able structural similarity (SI Text), the PvdRT-OpmQ efflux the periplasm of pvdRT-opmQ mutants, independent of the system does not provide any contribution to P. aeruginosa deleted component of the tripartite system (see Fig. 2), thus resistance to antibiotics belonging to the classes of tetracyclines,

Imperi et al. PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 ͉ 20443 Downloaded by guest on September 26, 2021 Fig. 5. PVD trafficking across the P. aeruginosa cell envelope. Ferribactin is synthesized in the cytoplasm and secreted by PvdE into the periplasm, where maturation to PVD is suggested to take place (1, 7). Export of de novo synthesized PVD occurs by a still uncharacterized mechanism. Ferri-PVD is translocated by FpvA into the periplasm, where Fe(III) is dissociated, plausibly by reduction to Fe(II) (14). Internalized PVD is recycled by the PvdRT-OpmQ system. It is hypothesized that Fe(II) enters the cytoplasm by a classic periplas- mic binding protein (PBP)/IM ABC importer (15). Experimentally confirmed and hypothetical steps are in yellow and gray, respectively.

On the other hand, comparable levels of extracellular PVD and no periplasmic PVD retention were observed in the PVD- producing uptake-mutant fpvA, irrespective of the pvdR muta- tion (see Fig. 4). These results lead us to propose that the PvdRT-OpmQ system is responsible for exogenous PVD recy- cling, while excluding its involvement in secretion of de novo synthesized PVD. Coherently, P. aeruginosa mutants with an impaired PvdRT-OpmQ system secrete up to 180 ␮M PVD in DCAA medium (see Fig. 1 and ref. 4), and our preliminary results suggest that the lower PVD levels in their culture supernatants are a result of reduced PVD synthesis rather than of impaired secretion of de novo synthesized PVD. A schematic of PVD trafficking in P. aeruginosa is shown in Fig. 5. What is the benefit for P. aeruginosa of having a system for Fig. 4. Effect of impaired PVD uptake on PVD accumulation in the periplasm. PVD recycling? The energetic cost of PVD synthesis is prohib- P. aeruginosa mutants were grown for 14 h at 37 °C in DCAA. (A) Quantifi- itive, and this is confirmed by the fact that PVD producers are cation of extracellular (black bars, Left y axis), periplasmic and spheroplasts- outcompeted by nonproducers in growth competition assays associated PVD (gray and white bars, respectively; Right y axis). Values are (21). Although PVD recycling does not provide a great advan- mean (Ϯ SD) of three independent assays. (B) Confocal microscopy images of tage to iron-deplete laboratory cultures, where the PVD con- cells. For each strain, the left and right panels show the visible and the centration attains up to 350 ␮M, circumstances may differ in corresponding 405-nm laser line confocal microscopy pictures, respectively. natural environments, such as the CF lung, where PVD is approximately 1 ␮M (22). Moreover, P. aeruginosa adopts a aminoglycosides, ␤-lactams and macrolides, and chloramphenicol biofilm mode of growth in the CF lung, which could further (Table S3). reduce the local availability of PVD. Notably, PVD-deficient Periplasmic accumulation of fluorescent PVD in P. aeruginosa mutants have been isolated from sputa of CF patients (23, 24), cells deficient in PvdRT-OpmQ could be explained by two indicating that P. aeruginosa occurs in the CF lung as a mixed community of PVD producers and PVD-deficient cheaters. In nonmutually exclusive mechanisms. According to the ‘‘de novo vitro and in vivo studies demonstrated that both growth and pathway,’’ PVD production could require PvdE-dependent virulence of these mixed populations are negatively affected by transport of the ferribactin precursor through the IM. Ferrib- increasing proportions of PVD cheaters over PVD producers actin would undergo maturation in the periplasm before extra- (reviewed in ref. 25). The ability to recycle PVD and, thus, to cellular release through a secretion device whose impairment make it available for virtually endless cycles of iron uptake, would cause periplasmic accumulation of PVD. In the ‘‘recycling would allow the P. aeruginosa community to maintain PVD pathway,’’ mature PVD would enter the periplasm by FpvA- levels at biologically meaningful concentrations with minor costs mediated uptake, where it would be retained in the absence of for the whole population. The biological relevance of an energy- a recycling system. saving device for PVD recycling is testified by the presence of To verify the involvement of PvdRT-OpmQ in either of these pvdRT-opmQ orthologs in the genome of all fluorescent pseudo- processes, accumulation of PVD by PvdRT-OpmQ-deficient monads sequenced so far (26) (Table S4). cells was assessed in PVD synthesis- or uptake-defective back- grounds. With respect to the biosynthetic (pvdA) mutant, the Materials and Methods pvdApvdR double mutant accumulated higher amounts of Growth Conditions. Bacterial strains and plasmids used in this study are listed exogenously-provided PVD in the periplasm (see Fig. 3 A–C), in Table S1. P. aeruginosa transposon insertion mutants were purchased from and was impaired in recycling of periplasmic PVD (see Fig. 3D). a P. aeruginosa Transposon Mutant Library (www.genome.washington.edu/

20444 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0908760106 Imperi et al. Downloaded by guest on September 26, 2021 UWGC/pseudomonas). P. aeruginosa was grown at 37 °C in DCAA medium bacterial cultures (OD600). Intracellular PVD was quantified fluorimetrically by (27), supplemented or not with 50 ␮M FeCl3 as iron-deplete and -replete recording the emission at 455 nm upon excitation at 398 nm in a LS50B conditions, respectively. Exogenous PVD was added as PVD-conditioned me- Luminescence spectrometer (Perkin–Elmer), using a standard curve generated dium (28), corresponding to a culture filtrate of the pyochelin-defective P. with known PVD concentrations (0–3 ␮M in 100 mM Tris-HCl, pH 8). aeruginosa PA6331 grown in DCAA medium. As a control, the medium con- ditioned by the PVD- and pyochelin-defective PA6331pvdA was used. Kinetics of PVD Uptake and Recycling. Kinetic studies of PVD uptake were performed by incubating 3 ϫ 109 cells/ml in the presence of 50 ␮M PVD in Construction of Deletion Mutants and Complementing Plasmids. E. coli was DCAA medium supplemented with 300 ␮g/ml kanamycin (Km) and 100 ␮g/ml routinely used for recombinant DNA manipulations. Site-specific excision of pvdR and pvdA genes was performed as previously described (29). For comple- chloramphenicol (Cm) to halt bacterial growth. At given time points, cells mentation of pvdR, pvdT, and opmQ mutations, a 4.7-kb fragment encom- from 1-ml samples were washed, lysed by sonication, and cell debris removed passing the pvdRT-opmQ operon with its putative promoter (region 2642032– by centrifugation for fluorimetric PVD measurements. 2646727 of the P. aeruginosa PAO1 chromosome, www.pseudomonas.com) PVD recycling studies were performed by incubating 2 ϫ 109 cells/ml with was amplified by PCR and cloned in pUCP19, yielding plasmid pUCPpvdRT- 1 ␮M ferri-PVD in DCAA medium supplemented with 300 ␮g/ml Km and 100 opmQ (see Table S1). ␮g/ml Cm. At given time points, 1-ml samples were centrifuged, and the appearance of apo-PVD was monitored fluorimetrically in both supernatants Subcellular Fractions. Intracellular PVD determinations were performed on P. and intact cells. aeruginosa cells grown for 14 h in DCAA, and washed three times with 30 mM ϫ 9 Tris-HCl (pH 7), 150 mM NaCl. Bacterial pellets (5 10 cells) were resuspended Confocal Microscopy. Cells (5 ϫ 108) from iron-deplete or -replete cultures were in 1 ml of 10 mM Tris-HCl (pH 8), 100 mM NaCl, and lysed by sonication. Cell washed and mounted onto agarose-coated slides as described (31). Images debris were removed by low-speed centrifugation (7,000 ϫ g, 10 min, 4 °C), were electronically acquired through a Leica SP5 confocal laser scanning and PVD concentration was determined using appropriate dilutions of cell microscope (Leica Microsystems) using a violet laser diode emitting at 405 nm. lysates in 100 mM Tris-HCl (pH 8). Periplasmic fractions were obtained by spheroplasting P. aeruginosa cells Laser intensity was kept constant at 11% for all images, without adjustments 9 of contrast and brightness. with lysozyme and MgCl2 as described (30). Briefly, bacterial pellets (5 ϫ 10 cells) were suspended in 1 ml of 10 mM Tris-HCl (pH 8.4), 200 mM MgCl2, 0.5 mg/ml lysozyme, and incubated for 30 min at room temperature with gentle Note Added in Proof. Since the work described in this paper was completed and shaking. Suspensions were centrifuged (11,000 ϫ g, 15 min, 4 °C) to collect the submitted for publication, the involvement of PvdE in ferribactin secretion periplasm-containing supernatants. The resulting pellets were suspended in 1 from the cytoplasm has been proposed by Yeterian E, et al. (32). ml of 10 mM Tris-HCl (pH 8), 100 mM NaCl, and lysed by sonication. Cell debris were removed to obtain the spheroplasts fractions. The specificity and purity ACKNOWLEDGMENTS. We thank Dr. F. Florenzano for assistance in confocal of cell fractions were verified by using specific subcellular protein markers as microscopy imaging of cells, and Prof. K. Poole for providing us with the P. described (30). aeruginosa tonB1 mutant. This work was supported by Grant PRIN-2006 from the Ministry of University and Research of Italy (to P.V.) and Grants FFC#10/ PVD Measurements. PVD from culture supernatants was diluted in 100 mM 2007 and FFC#8/2008 from the Italian Cystic Fibrosis Research Foundation Tris-HCl (pH 8), and measured as OD405 normalized by the cell density of (to P.V.).

1. Visca P, Imperi F, Lamont IL (2007) Pyoverdine siderophores: from biogenesis to 16. Takase H, Nitanai H, Hoshino K, Otani T (2000) Requirement of the Pseudomonas biosignificance. Trends Microbiol 15:22–30. aeruginosa tonB gene for high-affinity iron acquisition and infection. Infect Immun 2. Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML (2002) Siderophore-mediated 68:4498–4504. signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl 17. Elkins CA, Nikaido H (2002) Substrate specificity of the RND-type multidrug efflux Acad Sci USA 99:7072–7077. pumps AcrB and AcrD of Escherichia coli is determined predominantly by two large 3. Visca P (2004) in Pseudomonas, Volume 2, ed. Ramos JL (Kluwer Academic/Plenum). pp periplasmic loops. J Bacteriol 184:6490–6498. 69–123. 18. Mao W, et al. (2002) On the mechanism of substrate specificity by resistance nodulation 4. Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML (2002) GeneChip expression analysis of division (RND)-type multidrug resistance pumps: the large periplasmic loops of MexD from Pseudomonas aeruginosa are involved in substrate recognition. Mol Microbiol the iron starvation response in Pseudomonas aeruginosa: identification of novel 46:889–901. pyoverdine biosynthesis genes. Mol Microbiol 45:1277–1287. 19. Eda S, Maseda H, Nakae T (2003) An elegant means of self-protection in Gram-negative 5. Lewenza S, Gardy JL, Brinkman FS, Hancock RE (2005) Genome-wide identification of bacteria by recognizing and extruding xenobiotics from the periplasmic space. J Biol

Pseudomonas aeruginosa exported proteins using a consensus computational strategy MICROBIOLOGY Chem 278:2085–2088. combined with a laboratory-based PhoA fusion screen. Genome Res 15:321–329. 20. Yamanaka H, Kobayashi H, Takahashi E, Okamoto K (2008) MacAB is involved in the 6. Voulhoux R, Filloux A, Schalk IJ (2006) Pyoverdine-mediated iron uptake in Pseudo- secretion of Escherichia coli heat-stable enterotoxin II. J Bacteriol 190:7693–7698. monas aeruginosa: the Tat system is required for PvdN but not for FpvA transport. J 21. Griffin AS, West SA, Buckling A (2004) Cooperation and competition in pathogenic Bacteriol 188:3317–3323. bacteria. Nature 430:1024–1027. 7. Baysse C, Budzikiewicz H, Uria Fernandez D, Cornelis P (2002) Impaired maturation of 22. Haas B, Kraut J, Marks J, Zanker SC, Castignetti D (1991) Siderophore presence in sputa the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 of cystic fibrosis patients. Infect Immun 59:3997–4000. deficient for the cytochrome c biogenesis protein CcmC. FEBS Lett 523:23–28. 23. De Vos D, et al. (2001) Study of pyoverdine type and production by Pseudomonas aerugi- 8. Poole K, Heinrichs DE, Neshat S (1993) Cloning and sequence analysis of an EnvCD nosa isolated from cystic fibrosis patients: prevalence of type II pyoverdine isolates and homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement accumulation of pyoverdine-negative mutations. Arch Microbiol 175:384–388. in the secretion of the siderophore pyoverdine. Mol Microbiol 10:529–544. 24. Smith EE, et al. (2006) Genetic adaptation by Pseudomonas aeruginosa to the airways 9. McMorran BJ, Merriman ME, Rombel IT, Lamont IL (1996) Characterisation of the pvdE of cystic fibrosis patients. Proc Natl Acad Sci USA 103:8487–8492. gene which is required for pyoverdine synthesis in Pseudomonas aeruginosa. Gene 25. Buckling A, et al. (2007) Siderophore-mediated cooperation and virulence in Pseudo- 176:55–59. monas aeruginosa. FEMS Microbiol Ecol 62:135–141. 10. Poole K, Neshat S, Krebes K, Heinrichs DE (1993) Cloning and nucleotide sequence 26. Ravel J, Cornelis P (2003) Genomics of pyoverdine-mediated iron uptake in pseudo- analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa. J monads. Trends Microbiol 11:195–200. Bacteriol 175:4597–4604. 27. Visca P, Ciervo A, Sanfilippo V, Orsi N (1993) Iron-regulated salicylate synthesis by 11. Greenwald J, Zeder-Lutz G, Hagege A, Celia H, Pattus F (2008) The metal dependence Pseudomonas spp. J Gen Microbiol 139:1995–2001. 28. Banin E, Vasil ML, Greenberg EP (2005) Iron and Pseudomonas aeruginosa biofilm of pyoverdine interactions with its outer membrane receptor FpvA. J Bacteriol formation. Proc Natl Acad Sci USA 102:11076–11081. 190:6548–6558. 29. Imperi F, et al. (2008) Membrane-association determinants of the omega-amino acid 12. Ghysels B, et al. (2004) FpvB, an alternative type I ferripyoverdine receptor of Pseudo- monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aerugi- monas aeruginosa. Microbiology 150:1671–1680. nosa. Microbiology 154:2804–2813. 13. Schalk IJ, Abdallah MA, Pattus F (2002) Recycling of pyoverdin on the FpvA receptor 30. Imperi F, et al. (2009) Analysis of the periplasmic proteome of Pseudomonas aerugi- after ferric pyoverdin uptake and dissociation in Pseudomonas aeruginosa. Biochem- nosa, a metabolically versatile opportunistic . Proteomics 9:1901–1915. istry 41:1663–1671. 31. Mascarenhas J, Soppa J, Strunnikov AV, Graumann PL (2002) Cell cycle-dependent 14. Greenwald J, et al. (2007) Real time fluorescent resonance energy transfer visualization localization of two novel prokaryotic chromosome segregation and condensation of ferric pyoverdine uptake in Pseudomonas aeruginosa. A role for ferrous iron. J Biol proteins in Bacillus subtilis that interact with SMC protein. EMBO J 21:3108–3118. Chem 282:2987–2995. 32. Yeterian E, et al. (2009) Synthesis of the siderophore pyoverdine in Pseudomonas 15. Cornelis P (2008) Unexpected interaction of a siderophore with aluminum and its aeruginosa involves a periplasmic maturation. Amino Acids. 10.1007/s00726-009- receptor. J Bacteriol 190:6541–6543. 0358-0.

Imperi et al. PNAS ͉ December 1, 2009 ͉ vol. 106 ͉ no. 48 ͉ 20445 Downloaded by guest on September 26, 2021