Laboratory of Microbial Interactions, Department of Immunology, Parasitology and Ultrastructure, Flanders Interuniversity Institute of Biotechnology and Vrije Universiteit Brussel, Paardenstraat 65, B-1640 Sint Genesius Rode, Belgium1
University of Colorado Health Sciences Center, Microbiology, Box B-175, 4200 E Ninth Avenue, Denver, CO 80202, USA2
Laboratoire de Microbiologie et de Génétique, Université Louis Pasteur, UPRES-A 7010, F-67000 Strasbourg, France3
Institut für Organische Chemie der Universität zu Köln, Greinstrasse 4,D-50939 Köln, Germany4
Author for correspondence: Pierre Cornelis. Tel: +32 2 3590221. Fax: +32 2 3590390. e-mail: pcornel{at}vub.ac.be
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ABSTRACT |
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Keywords: Pseudomonas, vanadium, siderophores, oxidative stress
Abbreviations: CAS, chrome azurol; EDDHA, ethylenediamine-di(o-hydroxyphenylacetic acid); ESI, electrospray ion; PCH, pyochelin; PVD, pyoverdine; SOD, superoxide dismutase
a Present address: Brandwonden Centrum, Militair Hospitaal Koningin Astrid, B-1120 Brussels, Belgium.
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INTRODUCTION |
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Under conditions of iron limitation, Pseudomonas aeruginosa produces two siderophores, pyochelin (PCH), a thiazolin derivative (Cox et al., 1981 ), and pyoverdine (PVD), a fluorescent siderophore, which is composed of a chromophore (a quinoline derivative) and a peptide arm (Budzikiewicz, 1993
, 1997
). PVD has a much higher affinity for Fe(III) than PCH, which is consistent with PVD-negative mutants being unable to grow in the presence of the strong iron chelator ethylenediamine-di(o-hydroxyphenylacetic acid) (EDDHA) and being avirulent in a mouse infection model (Meyer et al., 1996
). The production of siderophores and of the proteins required for their uptake needs to be tightly regulated in order to avoid wastage of energy and accumulation of iron, which can be toxic for the cell because free
can generate toxic oxygen radicals via the Fenton reaction.
Iron uptake in P. aeruginosa is controlled at different levels via a complex interaction of regulators (Vasil & Ochsner, 1999 ). The ferric uptake regulator (Fur) is a general regulator, which, together with
as co-repressor, represses the transcription of the pvdS gene, which encodes a sigma factor needed for the transcription of PVD biosynthetic genes. Fur also controls the transcription of pchR, a gene that encodes an activator for the transcription of the PCH biosynthetic genes. Using an elegant approach, Ochsner & Vasil (1996)
found that Fur regulates a large array of genes in P. aeruginosa, including genes for putative siderophore receptors, haem uptake, alternative sigma factors, two-component regulatory systems, regulators and other unknown genes. Another gene repressed by Fur is sodA, which encodes the Mn-superoxide dismutase (SOD) of P. aeruginosa (Hassett et al., 1997a
). Two SODs are produced by P. aeruginosa, a Mn-SOD induced under low-iron conditions, and an Fe-SOD, which is the predominant form (Hassett et al., 1993
). These enzymes catalyse the dismutation of
to H2O2 (hydrogen peroxide) and O2; the peroxide, in turn, can be converted to H2O and O2 by catalases. Both reactive oxygen species are the product of the Fenton reaction catalysed by free
. Knowing that vanadium can substitute for iron in siderophores and in some iron-proteins such as transferrin (Keller et al., 1991
; Saponja & Vogel, 1996
), we investigated whether vanadyl ions could interfere with siderophore-mediated iron uptake systems and/or induce an oxidative stress in P. aeruginosa.
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METHODS |
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Detection and quantification of PVD in culture supernatants.
PVD was detected in culture supernatants by measuring the absorbance at 400 nm. The concentration of PVD was estimated spectrophotometrically at 400 nm using a molar absorption coefficient, 400, of 2 x 104 M-1 cm-1 (Höfte et al., 1993
) and normalized to the OD600 value of the culture.
To determine the effect of vanadium on PVD production, PAO1 cells were grown for 12 h in CAA containing 0, 0·5, 1·0 or 1·5 mM VOSO4 and then diluted to start a new culture in CAA for 48 h at 37 °C using the Bioscreen apparatus. Each experiment was performed in triplicate. The PVD content in the supernatant was determined by measuring A400.
Chrome azurol (CAS) detection of complex formation between siderophores and vanadium.
A vanadium/CAS solution was prepared by a method adapted from the original protocol described by Schwyn & Neilands (1987) . Six millilitres of 10 mM hexadecyltrimethylammonium bromide (HDTMA) was first diluted to 20 ml with water, to which 5·5 ml of 0·5 mM VOSO4.5H2O (in 10 mM HCl) was added. To this solution, 7·5 ml of 2 mM CAS was added slowly under constant agitation. Finally, 35 ml piperazine solution were added (4·3 g piperazine in 30 ml water and 5 ml pure HCl) and 5-sulfosalicylic acid to a final concentration of 4 mM. The final volume was adjusted to 100 ml. This purple vanadium/CAS solution could be poured as a gel after addition of 1% (w/v) agarose.
Siderophore and vanadiumsiderophore complex purification.
PVD (succinamide isoform) from P. aeruginosa PAO1 was isolated from CAA growth supernatants by the chloroform/phenol method followed by chromatography on CM-Sephadex and elution with 0·1 M pyrimidine/acetic acid buffer pH 5, as previously described (Hohnadel & Meyer, 1988 ). Eighty micromoles of the pure compound solubilized in 2 ml de-ionized water was supplemented with a slight excess of VOSO4.5H2O (100 µmol) and then filtered through a Sephadex G25 column (2·5 x 45 cm) eluted with de-ionized water. The major peak showing a brownish colour was collected and lyophilized to obtain the dark brown compound further analysed as a 1:1 VPVD complex.
PCH was extracted with chloroform from pH-3-acidified growth supernatants and purified by Sephadex-LH20 chromatography as described by Meyer et al. (1989) . Complexation with vanadium was achieved by mixing 90 µmol PCH in methanol solution with 100 µmol VOSO4.5H2O. The red-brown complex that formed instantaneously was purified by chromatography on Sephadex-LH20 (1·5 x 30 cm column, elution with methanol).
Mass spectrometric analysis of vanadiumsiderophore complexes.
Mass spectra were obtained with a Finnigan MAT (Bremen) 900 ST instrument equipped with an electrospray ion (ESI) source, an electrostatic and a magnetic analyser, and an ion trap system.
Plate assay for sensitivity to paraquat.
CAA or LB plates containing 0, 0·5 or 1·0 mM VOSO4.5H2O were inoculated with 107 cells (1 ml) from pre-cultures and dried for 1 h. Resistance to paraquat was analysed according to Hassett et al. (1995) .
SOD activity measurements.
Total SOD activity present in P. aeruginosa crude extracts was measured using the pyrogallol auto-oxidation inhibition assay described by Marklund & Marklund (1974) . Briefly, 30 µl pyrogallol (20 mM), 30 µl diethylenetriaminepentaacetic acid (DTPA; 0·1 M) and 100 µl freshly prepared crude cell extract (300 µg total protein) were added to 1·5 ml 0·1 M Tris/cacodylic acid pH 7·8; the final volume was adjusted to 3 ml with water. The change in A420 was monitored at 25 °C for 20 min. In the control auto-oxidation sample, the crude extract was replaced by 50 mM sodium phosphate buffer (cell lysis buffer; pH 7). P. aeruginosa cell-free extracts were prepared from overnight cultures in CAA or in CAA containing 0·5 or 1·0 mM VOSO4 as described by Clare et al. (1984)
except that, instead of using a French press, cells were lysed by sonication.
Construction of sodA and sodAsodB mutants of P. aeruginosa PAO1.
A 1150 bp fragment containing the sodA region was PCR-amplified using the primers 5'-GATGTGGCGCTGGAAAACAC and 5'-GCCAGTCGATCACGTTGTAG. The PCR product was cloned into pCRII-2.1 (Invitrogen), and a 1·7 kb gentamicin resistance (Gmr) cassette was cloned into the unique HincII site within the sodA gene. The 3·2 kb sodA::Gmr construct was excised from the pCRII-2.1 polylinker with PvuII and ligated into the SmaI site of the gene replacement vector pEX100T, which allows sacB counter-selection (Schweizer & Hoang, 1995 ). Escherichia coli SM10 containing pEX100T-sodA::Gmr was used as the donor strain in a bi-parental mating with P. aeruginosa PAO1. Transconjugants were selected on BHI agar containing gentamicin (75 µg ml-1) and irgasan (50 µg ml-1), and subsequently plated on LB agar containing gentamicin (75 µg ml-1) and 5% (w/v) sucrose. Successful double-crossover events resulting in sodA::Gmr mutants were monitored by the loss of a pEX100T-encoded carbenicillin resistance (Cbr) marker. The insertion of the Gmr cassette into sodA was confirmed by PCR across the sodA::Gmr region using the primers above. This yielded a 2·8 kb product for sodA::Gmr mutants compared to a 1·15 kb product for PAO1 wild-type (data not shown).
A 1380 bp PCR product containing the sodB region was amplified with primers 5'-TGATGGTGGCGGCCATGATG and 5'-ATCGCCATTTCCCGGTCGAG and ligated into pCRII-2.1. The PCR product was excised with HindIII and XbaI and cloned into pUC19. A 540 bp NcoIHincII fragment containing most of the sodB coding region was excised, the ends of the sodB flanking regions were filled in with Klenow enzyme and ligated to a 1·4 kb tetracycline resistance (Tcr) cassette which had been obtained by cutting pBR322 with EcoRI and StyI followed by end-polishing. The 2·6 kb sodB::Tcr construct was excised from the pUC19 polylinker with PvuII and ligated into the SmaI site of pEX100T. E. coli SM10 harbouring the resulting plasmid, pEX100T-
sodB::Tcr, was used as the donor strain in a biparental mating with P. aeruginosa sodA::Gmr. The mating and the subsequent isolation of sodAsodB mutants were performed under anaerobic conditions using Campy Pak jars with palladium catalyst (Becton-Dickinson) in the presence of 1% (w/v) potassium nitrate as an alternative electron acceptor during anaerobic growth. Candidate sodA::Gmr sodB::Tcr double mutants were initially selected on BHI agar containing 1% (w/v) KNO3, tetracycline (150 µg ml-1) and irgasan (50 µg ml-1), followed by sacB counter-selection on LB agar containing 5% sucrose, 1% KNO3 and tetracycline (150 µg ml-1). The successful replacement of the sodB gene by the Tcr cassette was verified by PCR across the sodB region using the above primers, resulting in a 2·2 kb PCR product for the sodA::Gmr sodB::Tcr double mutant compared to a 1·38 kb product for the sodA::Gmr single mutant (data not shown).
PCR was performed using Taq polymerase and custom-made primers (Bethesda Research Laboratories) in a Perkin-Elmer Cetus thermal cycler, with 30 cycles of denaturing (1 min, 94 °C), annealing (1 min, 54 °C) and extending (1 min per kb of DNA, 72 °C). The PCR products were purified in low-melting-point agarose gels, routinely cloned into pCRII-2·1 (Invitrogen) and sequenced with Sequenase 2·0 (United States Biochemical) and M13 primers or custom-made 18-mer oligonucleotides.
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RESULTS |
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Inhibition of growth of wild-type PAO1 and siderophore-negative mutants by vanadium
Fig. 1 shows the growth curves of wild-type PAO1 and two PVD-negative mutants. These mutants are non-fluorescent under UV, do not produce PVD and are unable to grow in the presence of the Fe(III) chelator EDDHA. These two mutants grew in the presence of 1 mM vanadium only after a prolonged lag phase. Conversely, all mutants defective in the production of PCH showed an increased resistance to vanadium, compared to the wild-type, although to different extents (Fig. 2
). The two PVD+ PCH- mutants especially were found to be completely unaffected by the presence of 1·5 mM VOSO4, a concentration that severely compromised the growth of the wild-type strain. The PCH- Sal+ and the PCH- Sal- mutants showed an intermediate level of resistance to vanadium. These results led us to conclude that the production of PVD contributes to resistance to vanadium whilst, conversely, the production of PCH enhances the toxic effect of vanadium.
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Vanadium complexation by PVD and PCH
Spent medium of PVD-producing strains grown in the presence of vanadium showed a characteristic brown colour that was not observed in spent medium of PVD-negative mutants. Furthermore, the fluorescence under UV, characteristic of free PVD, could not be detected in cultures of P. aeruginosa grown in the presence of 0·5 mM VOSO4. This prompted us to investigate whether PVD and PCH could complex vanadium. For both purified siderophores, a very clear shift of the UV-visible peaks was observed after addition of 1 mM VOSO4, an indication that a complex was formed. The following peaks were observed: purified free PVD at pH 5·2, 365 and 385 nm; PVD plus VOSO4, 405 nm; purified PCH in methanol, 218 and 248 nm; PCH plus VOSO4, 232 and 273 nm. Using another approach to detect vanadium binding by both siderophores, a vanadium-CAS assay was developed whereby iron was replaced by vanadium. Addition of both siderophores to a vanadium/CAS plate resulted in a rapid colour change due to the de-complexation of vanadiumCAS (Fig. 3).
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The molecular ion region of the PVD (succinamide side chain)V complex showed two peaks, viz. m/z 1397, corresponding to [PVD-2H++VO+] + H+ and m/z 1429, corresponding to [PVD-2H++VO2++CH3OH] + H+. This is in agreement with the replacement of two H+ by V(0). V(IV)O2+ has only four co-ordination sites free to accommodate two of the three bidentate ligands of PVD (catecholate and two N-formyl-N-hydroxy-Orn). One site is occupied by the oxygen atom. To complete the octahedral structure, the remaining site binds one molecule of CH3OH, the solvent used for the ESI measurements. In an aqueous medium it is probably replaced by H2O. That the mass difference of 32 units is actually due to CH3OH (and not, for example, to O2) was confirmed by a determination of the exact mass difference between the two ions.
The PCHV complex solution contains mainly (approx. 85%) ions with the composition [PCH-2H++VO2+] + K+ (m/z 428) (form 1). Again V(IV)O2+ replaces two H+. The neutral complex 1 is then ionized by attachment of K+. The isotope pattern confirms the composition. A second component (approx. 9%) corresponds according to its exact mass and isotope pattern to [PCH-2H++VO2+]- + K+ (m/z 483) (form 2). In this case V(V) has been incorporated. The negative complex 2 needs the attachment of two K+ to give a positive ion in the mass spectrometer. The last approximately 6% consisted of the uncomplexed anion of PCH, [PCH-H+]- + 2K+ (m/z 401) (form 3), again confirmed by exact mass measurements and the isotope pattern. As frequently observed in ESI mass spectrometry, cluster ions were also seen, such as [1+3]-+ 2K+ (m/z 790), [1+1] + K+ (m/z 817), [1+2]- + 2K+ (m/z 872), [1+1+1] + K+ (m/z 1206) and [1+1+2]- + K+ (m/z 1261).
Effect of VPVD and VPCH complexes on growth of P. aeruginosa PAO1
Purified vanadyl-siderophores were tested for their inhibitory effect on growth of P. aeruginosa PAO1 in CAA medium. VPVD up to 125 µg ml-1 did not affect the growth of PAO1, whilst, conversely, VPCH had a very striking inhibitory effect on growth (Fig. 4). Addition of VPCH resulted in a prolongation of the lag phase that lasted for more than 50 h before growth resumed.
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Effect of mutations in sodA and sodB genes on the resistance of P. aeruginosa to vanadium
P. aeruginosa produces two SODs, one Mn-co-factored (SodA), and another Fe-co-factored (SodB) (Hassett et al., 1993 ). As already mentioned, resistance to vanadium was higher in the presence of iron (Table 2
), indicating that SodB could be the major contributor to the resistance since the production of this haem-containing enzyme was shown to be increased under conditions of iron sufficiency (Hassett et al., 1993
). Furthermore, it is known that SodB contributes more to resistance to superoxide induced by paraquat than SodA (Hasset et al., 1995
).
SOD mutants from PAO1 and a cystic fibrosis strain, FRD1 (Hassett et al., 1997b ), were grown in CAA or in CAA containing 1 mM VOSO4. The results are shown in Fig. 5(a)
for strain PAO1 (wild-type, and sodA and sodAsodB mutants), and in Fig. 5(b)
for strain FRD1 (wild-type, and sodA, sodB and sodAsodB mutants). The growth of both sodA mutants (PAO1 and FRD1) was relatively unaffected by the presence of 1 mM VOSO4 compared to the respective wild-type strains. However, the growth of the sodB (FRD1) mutant was strongly inhibited in the presence of vanadium. The double sodAsodB mutants grew either very poorly (in the case of FRD1) or not at all (PAO1). These results again indicated that the resistance to vanadium-generated superoxides is largely due to SodB.
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DISCUSSION |
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The inhibitory effect of vanadium was strongly enhanced by the presence of non-utilizable iron(III) chelators such as EDDHA or a heterologous, non-cognate PVD. This could mean that vanadium interferes with siderophore-mediated iron uptake in P. aeruginosa. One way by which vanadium could affect iron uptake is via the formation of stable vanadyl or vanadatesiderophore complexes that, eventually, could be taken up by the cell, increasing the intracellular concentration of vanadium. Alternatively, the vanadiumsiderophore complexes could block the uptake of ironsiderophores in a competitive way. The competition of vanadium with iron for binding by siderophores could explain why the uptake of iron via the siderophores is compromised. However, we do not at this stage know whether the vanadylsiderophores are actively taken up by the cells.
We demonstrate here that vanadium can indeed form complexes with the two siderophores of P. aeruginosa, PCH and PVD. This is not surprising since it is well established that vanadium can be liganded by hydroxamates, carboxylates and catecholates (Keller et al., 1991 ; Rehder, 1991
). Others have also demonstrated the fact that PVD, as well as PCH, can complex copper, zinc or manganese (Poppe et al., 1987
; Chen et al., 1994
; Visca et al., 1992
; Bouby et al., 1999
). Visca et al. (1992)
suggested that PCH could play a role in the acquisition of metals other than Fe(III), such as Co(II) and Mo(VI). Likewise, azotochelin, a catecholate siderophore, has been suggested to participate in the uptake of molybdenum by Azotobacter vinelandii (Duhme et al., 1998
).
Here we show that the VPCH complex, but not the VPVD complex, has a strong antibacterial effect. Such an effect was not observed by Visca et al. (1992) for PCH complexed with other metals, including Mo(VI) and Cu(II). Interestingly, two forms of VPCH complexes were found, one with V(IV) and one with V(V). This result suggests that VPCH can undergo an oxidative cycle that can result in the generation of superoxide radicals (
). It is well known that ferripyochelin can undergo such a redox cycle, resulting in the production of cell-damaging active oxygen species (Coffman et al., 1990
; Britigan et al., 1997
). Stern et al. (1992)
also demonstrated that V(IV)desferrioxamine has a redox-cycling activity and generates active oxygen species. In another study on A. vinelandii siderophores, Cornish & Page (1998)
demonstrated that, among the three catecholate siderophores produced by this bacterium, only aminochelin, which has the lowest affinity for iron(III), was unable to sequester iron and prevent a Fenton reaction. Interestingly, the same authors also observed that increased aeration resulted in a parallel increase in the production all three catecholate siderophores of A. vinelandii, aminochelin, azotochelin and protochelin. Protochelin is a tri-catecholate with the highest affinity for iron and is produced only under conditions of extreme oxygen stress. Like protochelin, PVD from P. aeruginosa can form a 1:1 complex with iron and protect the cells from oxidative damage due to the Fenton reaction (Coffman et al., 1990
). Indeed, these authors showed that
could reduce and release iron bound to FePCH but not FePVD.
The fact that PVD-negative mutants are more affected by vanadium can be explained by their higher production of PCH (Höfte et al., 1993 ), which results in an increase in the toxicity of vanadium.
Another intriguing observation we report is the repression of PVD production when wild-type cells were grown in the presence of vanadium. We can assume that vanadium did not interfere with the normal Fur-mediated regulation since iron-repressed outer-membrane proteins are normally produced in CAA medium containing vanadium and their production is repressed when iron is also present (results not shown).
We propose that VPCH molecules undergo an oxidative cycle, resulting in the production of reactive oxygen species that, in turn, induce a response in the form of increased SOD activity. We observed that a pre-incubation with vanadium salts increases the resistance of PAO1 cells both to vanadium and to the redox-cycling agent paraquat, and causes an increase in SOD activity. Analysis of sod mutants indicated that Mn-SOD, encoded by sodA, only marginally participates in the resistance towards vanadium in iron-limiting conditions, whereas SodB plays a major role. SodA is known to be regulated by the Fur repressor (Hassett et al., 1996 , 1997a
), whilst not much is known about the regulation of SodB production in P. aeruginosa except that its production is optimal under iron-sufficient conditions (Hassett et al., 1992
). The pronounced growth inhibitory effect of vanadium on iron-starved cells could be the result of insufficient production of the iron-containing superoxide-detoxifying enzyme SodB under these conditions. Further studies should include experiments designed to investigate whether vanadium uptake by the cells is increased by siderophores. Also, the search for vanadium-resistant or vanadium-susceptible mutants in P. aeruginosa should provide useful information about the mechanisms involved in the homeostasis of this metal, and the interaction between siderophores and oxidative stress in this bacterium.
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ACKNOWLEDGEMENTS |
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Received 23 March 2000;
revised 20 June 2000;
accepted 29 June 2000.