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Microbiology (2000), 146, 2425-2434.

Pseudomonas: Biology and Diversity

Vanadium interferes  with siderophore-mediated iron uptake  in Pseudomonas aeruginosa

Christine Baysse¹, Daniel De Vos^a,1, Yann Naudet¹, Alain Vandermonde¹, Urs Ochsner², Jean-Marie Meyer³, Herbert Budzikiewicz⁴, Matthias Schäfer⁴, Regine Fuchs⁴ and Pierre Cornelis¹
 

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@vub.ac.be

 
Vanadium is a metal that under physiological conditions can exist in two oxidation states, V(IV) (vanadyl ion) and V(V) (vanadate ion). Here, it was demonstrated that both ions can form complexes with siderophores. Pseudomonas aeruginosa produces two siderophores under iron-limiting conditions, pyoverdine (PVD) and pyochelin (PCH). Vanadyl sulfate, at a concentration of 1-2 mM, strongly inhibited growth of P. aeruginosa PAO1, especially under conditions of severe iron limitation imposed by the presence of non-utilizable Fe(III) chelators. PVD-deficient mutants were more sensitive to vanadium than the wild-type, but addition of PVD did not stimulate their growth. Conversely, PCH-negative mutants were more resistant to vanadium than the wild-type strain. Both siderophores could bind and form complexes with vanadium after incubation with vanadyl sulfate (1:1, in the case of PVD; 2:1, in the case of PCH). Although only one complex with PVD, V(IV)-PVD, was found, both V(IV)- and V(V)-PCH were detected. V-PCH, but not V-PVD, caused strong growth reduction, resulting in a prolonged lag phase. Exposure of PAO1 cells to vanadium induced resistance to the superoxide-generating compound paraquat, and conversely, exposure to paraquat increased resistance to V(IV). Superoxide dismutase (SOD) activity of cells grown in the presence of V(IV) was augmented by a factor of two. Mutants deficient in the production of Fe-SOD (SodB) were particularly sensitive to vanadium, whilst sodA mutants deficient for Mn-SOD were only marginally affected. In conclusion, it is suggested that V-PCH catalyses a Fenton-type reaction whereby the toxic superoxide anion is generated, and that vanadium compromises PVD utilization.

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.

 
Vanadium is a transition metal which, at neutral pH, can exist in two oxidation states, V(IV) (vanadyl ion; cationic species, ), and V(V) (vanadate ion; anionic species, H₂) (Rehder, 1991 , 1992 ). As a result, vanadium can affect diverse biological processes. Vanadate resembles phosphate (), and consequently can take its place in phosphate-metabolizing enzymes (Rehder, 1991 , 1992 ). Another important biological characteristic of both vanadate and vanadyl ions is that they can form complexes with carboxylate, catecholate and hydroxamate ligands, which are present in different siderophores (Keller et al., 1991 ; Rehder, 1991 ). Although this last characteristic is well known, surprisingly, there has been no study done so far on the influence of vanadate or vanadyl ions on the siderophore-mediated iron(III) uptake systems in Gram-negative bacteria. Such an investigation is justified if one considers the importance of siderophores for iron acquisition by pathogenic bacteria (Crosa, 1997 ). Furthermore, it is known that, at neutral pH, V(IV) can be readily oxidized to V(V), generating the toxic superoxide anion () (Liochev & Fridovich, 1987 ). It has also been shown that complexation of vanadium by the siderophore desferrioxamine enhances its redox cycling capacity in vitro (Stern et al., 1992 ). Despite this, the antibacterial activity of vanadium-siderophore complexes has never been investigated.

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.

 
Strains and growth conditions.
The P. aeruginosa strains used in this study are listed in Table 1. Cultures were grown in Luria-Bertani medium (LB) or in the iron-poor Casamino-acids medium (CAA) (Cornelis et al., 1992 ). Cultures were grown in a Bioscreen apparatus (Life Technologies) using the following parameters: shaking for 10 s every 3 min; reading every 20 min; temperature, 37 °C; volume of culture, 300 µl. As inoculum, an overnight culture of PAO1 in CAA was diluted in order to achieve a final optical density at 600 nm of 0·0001. Each culture was replicated three times and each experiment was performed in triplicate.

 
Vanadyl sulfate (VOSO₄.5H₂O) was prepared as a 100 mM stock solution and kept at 4 °C. Filter-sterilized FeCl₃ (0·1 M in 0·1 M HCl) was added to the medium after autoclaving at the concentrations indicated in the text. EDDHA was prepared as a 100 mg ml^-1 solution (pH adjusted to 9) and filter-sterilized.

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 vanadium-siderophore 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 V-PVD 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 vanadium-siderophore 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 (Gm^r) cassette was cloned into the unique HincII site within the sodA gene. The 3·2 kb sodA::Gm^r 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::Gm^r 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::Gm^r mutants were monitored by the loss of a pEX100T-encoded carbenicillin resistance (Cb^r) marker. The insertion of the Gm^r cassette into sodA was confirmed by PCR across the sodA::Gm^r region using the primers above. This yielded a 2·8 kb product for sodA::Gm^r 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 NcoI-HincII 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 (Tc^r) cassette which had been obtained by cutting pBR322 with EcoRI and StyI followed by end-polishing. The 2·6 kb sodB::Tc^r 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::Tc^r, was used as the donor strain in a biparental mating with P. aeruginosa sodA::Gm^r. 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::Gm^r sodB::Tc^r 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 Tc^r cassette was verified by PCR across the sodB region using the above primers, resulting in a 2·2 kb PCR product for the sodA::Gm^r sodB::Tc^r double mutant compared to a 1·38 kb product for the sodA::Gm^r 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.

 
Effect of iron on the growth of P. aeruginosa PAO1 in the presence of vanadium
Table 2 shows the effect of the addition of increasing concentrations of VOSO₄ (1·0, 1·5 and 2 mM) on the growth of PAO1 under iron-limiting or iron-sufficient conditions. The following parameters were analysed: the duration of the lag phase, the slope of the exponential phase (OD₆₀₀ min^-1), the maximal optical density (OD₆₀₀ max) reached, the time to reach the maximal OD₆₀₀, and the OD₆₀₀ after 48 h of growth. Addition of 10 µM FeCl₃ to the CAA medium resulted, as expected, in strong stimulation of growth as judged by the maximal OD₆₀₀ reached and the increase in the slope. When iron was present, the addition of increasing concentrations of VOSO₄ resulted in a concentration-dependent increase in the lag phase (up to 35 h for the highest concentration of 2 mM), but once growth resumed, both the slope and the maximal OD₆₀₀ reached were relatively unchanged. In the absence of iron, increased concentrations of VOSO₄ not only caused a prolongation of the lag phase, but also decreased the slope and decreased the maximal OD₆₀₀. In the presence of 2 mM VOSO₄, no growth was observed in CAA medium without added iron, even after 72 h.

 
Effect of Fe(III) chelators on the growth of P. aeruginosa in the presence of vanadium
The effects of adding the strong, non-metabolizable Fe(III) chelator EDDHA, the cognate purified PVD, and a heterologous PVD from P. aeruginosa ATCC 27853 for which PAO1 has no receptor (Hohnadel & Meyer, 1988 ), on the growth of PAO1 in CAA medium was tested after 24 h. As expected, addition of the cognate PVD (50 µM) in CAA medium stimulated growth (0·49 OD₆₀₀ units compared with 0·36 OD₆₀₀ units for the CAA control), whilst addition of EDDHA (0·5 mg ml^-1) or the heterologous PVD (50 µM) had little effect on growth (0·29 and 0·40 OD₆₀₀ units, respectively). In the presence of a subinhibitory concentration of vanadium (1 mM), the OD₆₀₀ after 24 h (0·33 OD₆₀₀ units) was reduced compared to the CAA control. Strikingly, the combination of 1 mM vanadium and EDDHA resulted in complete suppression of growth (0·09 OD₆₀₀ units), an effect which persisted even when incubation was extended to 48 h (results not shown). Interestingly, addition of the cognate PVD in the presence of 1 mM VOSO₄ did not stimulate growth (0·22 OD₆₀₀ units), whilst addition of the non-cognate PVD, as in the case of EDDHA, resulted in complete growth suppression (0·09 OD₆₀₀ units).

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.

 
Effect of vanadium on PVD production
We wanted to investigate if pre-incubation of the cells with vanadium would affect PVD production since we had observed that the characteristic fluorescence due to PVD production disappeared when cells were grown in CAA medium containing 0·5 mM VOSO₄. Compared to the control condition (CAA medium, no VOSO₄, 100% PVD production), pre-culturing in the presence of 0·5, 1·0 and 1·5 mM VOSO₄ for 12 h caused a reduction in PVD production in CAA medium of 20, 40 and 42%, respectively, whilst cell growth, as determined by OD₆₀₀, was identical under each condition used. Conversely, PCH production was not affected by pre-exposure of the cells to 0·5 mM VOSO₄. The PCH content of supernatants from cultures of the PVD^- mutant 6609 grown in CAA was determined by liquid CAS assay (Schwyn & Neilands, 1987 ) and was found to be identical whether the pre-culture was performed in the presence or in the absence of 0·5 mM VOSO₄.

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 vanadium-CAS (Fig. 3).

 
Mass spectrometric analysis of the vanadium-siderophore complexes
After binding with vanadium, the PCH- and PVD-V complexes were purified as described in Methods and analysed by ESI-MS. In the case of PVD, a 1:1 complex with V(IV) was demonstrated, whilst in the case of PCH, a 2 PCH:1 V complex was found. Interestingly, for PCH, both V(IV) and V(V) were detected in the complexes.

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 PCH-V 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 V-PVD and V-PCH 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. V-PVD up to 125 µg ml^-1 did not affect the growth of PAO1, whilst, conversely, V-PCH had a very striking inhibitory effect on growth (Fig. 4). Addition of V-PCH resulted in a prolongation of the lag phase that lasted for more than 50 h before growth resumed.

 
Effect of vanadium on resistance of P. aeruginosa to paraquat
The strong inhibitory activity of V-PCH, and the fact that both V(IV) and V(V) were detected in association with PCH, were strongly suggestive of V-PCH undergoing a redox cycle which could result in the generation of toxic superoxide anions (), as already described for ferripyochelin (Coffman et al., 1990 ; Britigan et al., 1997 ). On the other hand, it is well known that V(IV) can auto-oxidize in neutral aqueous solutions, yielding and vanadate (Liochev & Fridovich, 1987 ). Cells grown in the presence of VOSO₄ were found to be more resistant to the superoxide-generating agent paraquat, as expressed by the decrease in the diameter of the growth inhibition zone caused by paraquat on CAA or LB plates (Table 3). This resistance increased as a function of the concentration of vanadium present in the medium. Pre-incubation of the cells with 0·5 mM VOSO₄ also caused a decrease in the growth inhibition caused by paraquat. In addition, we noticed that on CAA plates containing 1 mM VOSO₄ a ring of increased growth was visible at the limit of the paraquat growth inhibition zone (not shown). Altogether, these results strongly suggest that vanadium can induce an oxidative response and a defence mechanism in P. aeruginosa. Since both paraquat and vanadyl ions can generate superoxides, it is most likely that vanadyl ions can induce a SOD activity in P. aeruginosa. To test this hypothesis, total SOD activity was measured by the inhibition of the auto-oxidation of pyrogallol in crude extracts of P. aeruginosa PAO1 cells before and after pre-incubation with 0·5 mM VOSO₄. The total SOD activity in the conditions used was 10% before and 20% after exposure to VOSO₄ (expressed as percentage of inhibition of pyrogallol auto-oxidation). Such a doubling of SOD activity was consistently observed, confirming that vanadyl ions indeed induce an oxidative stress and an adaptive response in P. aeruginosa.

 
Pre-exposure of bacterial cells to subinhibitory vanadium concentrations induced a higher level of resistance, resulting in an increased growth rate, but only when iron was present during the pre-culture and the culture (Table 4). Similar results were obtained in LB medium (results not shown).

 
We also observed that PAO1 cells plated on P-agar containing 1 mM VOSO₄ produced more pyocyanin than on P-agar alone, an indication that the level of Fe-SOD might be elevated in the cells exposed to vanadium (Hassett et al., 1995 ).

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.

 
One of the main concerns for the treatment of bacterial infections is the increasing proportion of antibiotic-resistant strains. Metals, such as silver and cerium, are already used in topical applications because of their antibacterial activity (Klasen, 2000 ). Vanadium salts are known to exert, among others, anti-diabetic effects (Srivastava, 2000 ). Few reports mention the antibacterial effects of vanadium salts. In the case of Streptococcus pneumoniae, both vanadate and vanadyl compounds were found to have a strong antibacterial activity (Fukuda & Yamase, 1997 ). Although these authors found that vanadium salts had a negligible activity towards other bacteria, including Staphylococcus aureus, E. coli and P. aeruginosa, it is important to mention the fact that they grew these bacteria only in the iron-rich Mueller-Hinton medium. In this study, we demonstrate that vanadium salts inhibit the growth of P. aeruginosa, especially in an iron-limited medium. In an iron-replete medium, we observed that vanadium caused a prolongation of the lag phase without affecting the exponential phase. Conversely, under conditions of iron limitation, the lag phase was less affected by the presence of vanadium in the medium whilst the growth rate was severely reduced. Although we do not at present have an explanation for this phenomenon, it suggests that vanadium can affect different functions depending on the iron content of the cell.

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 vanadate-siderophore complexes that, eventually, could be taken up by the cell, increasing the intracellular concentration of vanadium. Alternatively, the vanadium-siderophore complexes could block the uptake of iron-siderophores 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 vanadyl-siderophores 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 V-PCH complex, but not the V-PVD 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 V-PCH complexes were found, one with V(IV) and one with V(V). This result suggests that V-PCH 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 Fe-PCH but not Fe-PVD.

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 V-PCH 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.

 
We thank Dr Cornelia Reimman for sending us the PCH mutants, and Dr Dan Hassett for sending us the FRD1 strain and the different sod mutants as well as for interesting discussions. We also thank Dr Theresa Pattery for carefully reading this manuscript. The Bioscreen apparatus was acquired thanks to the FWO (krediet aan navorser). This work was also supported by the Jean and Alphonse Forton Fund.

 
Bouby, M., Billard, I. & Maccordick, H. J. (1999). Selective behavior of the siderophore pyoverdine A towards , Th⁴⁺, U⁴⁺ and other cations. Czech J Phys 49, Suppl. 1, 147-150.

Britigan, B. E., Rasmussen, G. T. & Cox, C. D. (1997). Augmentation of oxidant injury to human pulmonary epithelial cells by the Pseudomonas aeruginosa siderophore pyochelin. Infect Immun 65, 1071-1076.

Budzikiewicz, H. (1993). Secondary metabolites from fluorescent pseudomonads. FEMS Microbiol Rev 104, 209-228.

Budzikiewicz, H. (1997). Siderophores of fluorescent pseudomonads. Z Naturforsch 52C, 713-720.

Chen, Y., Jurkevitch, E., Bar-Ness, E. & Hadar, Y. (1994). Stability constant of pseudobactin complexes with transition metals. Soil Sci Soc Am J 58, 390-396.

Clare, D. A., Duong, M. N., Darr, D., Archibald, F. & Fridovich, I. (1984). Effects of molecular oxygen on detection of superoxide radical with nitroblue tetrazolium and on activity stains for catalase. Anal Biochem 140, 532-537.

Coffman, T. J., Cox, C. D., Edeker, B. L. & Britigan, B. E. (1990). Possible role of bacterial siderophores in inflammation. J Clin Invest 86, 1030-1037.

Cornelis, P., Anjaiah, V., Koedam, N., Delfosse, P., Jacques, P., Thonart, P. & Neirinckx, L. (1992). Stability, frequency and multiplicity of transposon insertions in the pyoverdine region in the chromosome of different fluorescent pseudomonads. J Gen Microbiol 138, 1337-1343.

Cornish, A. S. & Page, W. J. (1998). The catecholate siderophores of Azotobacter vinelandii: their affinity for iron and role in oxygen stress management. Microbiology 144, 1747-1754.

Cox, C. D., Rinehart, K. L., Moore, M. L. & Cook, J. C. (1981). Pyochelin: novel structure of an iron chelating growth promoter for Pseudomonas aeruginosa. Proc Natl Acad Sci U S A 78, 4256-4260.

Crosa, J. H. (1997). Signal transduction and transcriptional and postranscriptional control of iron-regulated genes in bacteria. Microbiol Mol Biol Rev 61, 319-336.

Duhme, A. K., Hider, R. C., Naldrett, M. J. & Pau, R. N. (1998). The stability of the molybdenum-azotochelin complex and its effect on siderophore production in Azotobacter vinelandii. J Biol Inorg Chem 3, 520-526.

Fukuda, N. & Yamase, T. (1997). In vitro antibacterial activity of vanadate and vanadyl compounds against Streptococcus pneumoniae. Biol Pharm Bull 20, 927-930.

Goldberg, J. B. & Ohman, D. E. (1984). Cloning and expression in Pseudomonas aeruginosa of a gene involved in the production of alginate. J Bacteriol 158, 1115-1121.

Hassett, D. J., Charniga, L., Bean, K. A., Ohman, D. E. & Cohen, M. S. (1992). Response of Pseudomonas aeruginosa to pyocyanin: mechanisms of resistance, antioxidant defenses, and demonstration of a manganese-cofactored superoxide dismutase. Infect Immun 60, 328-336.

Hassett, D. J., Woodruff, W. A., Wozniak, D. J., Vasil, M. L., Cohen, M. S. & Ohman, D. E. (1993). Cloning and characterization of the Pseudomonas aeruginosa sodA and sodB genes encoding manganese- and iron-cofactored superoxide dismutase: demonstration of increased manganese-superoxide dismutase activity in alginate-producing bacteria. J Bacteriol 175, 7658-7665.

Hassett, D. J., Schweizer, H. P. & Ohman, D. E. (1995). Pseudomonas aeruginosa sodA and sodB mutants defective in manganese- and iron-cofactored superoxide dismutase activity demonstrate the importance of the iron-cofactored form in aerobic metabolism. J Bacteriol 177, 6330-6337.

Hassett, D. J., Sokol, P. A., Howell, M. L., Ma, J. F., Schweizer, H. T., Ochsner, U. A. & Vasil, M. L. (1996). Ferric uptake regulator (Fur) mutants of Pseudomonas aeruginosa demonstrate defective siderophore-mediated iron uptake, altered aerobic growth, and decreased superoxide dismutase and catalase activities. J Bacteriol 178, 3996-4003.

Hassett, D. J., Howell, M. L., Ochsner, U. A., Vasil, M. L., Johnson, Z. & Dean, G. E. (1997a). An operon containing fumC and sodA encoding fumarase C and manganese superoxide dismutase is controlled by the ferric uptake regulator in Pseudomonas aeruginosa: fur mutants produce elevated alginate levels. J Bacteriol 179, 1452-1459.

Hassett, D. J., Howell, M. L., Sokol, P. A., Vasil, M. L. & Dean, G. E. (1997b). Fumarase C activity is elevated in response to iron deprivation and in mucoid, alginate-producing Pseudomonas aeruginosa: cloning and characterization of fumC and purification of native FumC. J Bacteriol 179, 1442-1451.

Höfte, M., Buysens, S., Koedam, N. & Cornelis, P. (1993). Zinc affects siderophore-mediated high affinity iron uptake systems in the rhizosphere Pseudomonas aeruginosa 7NSK2. Biometals 6, 85-91.

Hohnadel, D. & Meyer, J. M. (1988). Specificity of pyoverdine-mediated iron uptake among fluorescent Pseudomonas. J Bacteriol 170, 4865-4873.

Hohnadel, D., Haas, D. & Meyer, J. M. (1986). Mapping of mutations affecting pyoverdine production in Pseudomonas aeruginosa. FEMS Microbiol Lett 36, 195-199.

Keller, R. J., Rush, J. D. & Grover, T. A. (1991). Spectrophotometric and ESR evidence for vanadium(IV) deferioxamine complexes. J Inorg Biochem 41, 269-276.

Klasen, H. J. (2000). A historical review of the use of silver in the treatment of burns. II. Renewed interest for silver. Burns 26, 131-138.

Liochev, S. & Fridovich, I. (1987). The oxidation of NADH by tetravalent vanadium. Arch Biochem Biophys 255, 274-278.

Marklund, S. & Marklund, G. (1974). Involvement of the superoxide anion radical in the autooxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur J Biochem 47, 469-474.

Meyer, J.-M., Hohnadel, D. & Hallé, F. (1989). Cepabactin from Pseudomonas cepacia, a new type of siderophore. J Gen Microbiol 135, 1479-1487.

Meyer, J.-M., Neely, A., Stintzi, A., Georges, C. & Holder, I. A. (1996). Pyoverdine is essential for virulence of Pseudomonas aeruginosa. Infect Immun 64, 518-523.

Meyer, J.-M., Stintzi, A., De Vos, D., Cornelis, P., Tappe, R., Taraz, K. & Budzikiewicz, H. (1997). Use of siderophores to type pseudomonads: the three Pseudomonas aeruginosa pyoverdine systems. Microbiology 143, 35-43.

Ochsner, U. A. & Vasil, M. L. (1996). Gene repression by the ferric uptake regulator in Pseudomonas aeruginosa: cycle selection of iron-regulated genes. Proc Natl Acad Sci U S A 93, 4409-4414.

Poppe, K., Taraz, K. & Budzikiewicz, H. (1987). Pyoverdine type siderophores from Pseudomonas fluorescens. T etrahedron 43, 2261-2272.

Rehder, D. (1991). The bioorganic chemistry of vanadium. Angew Chem 30, 148-167.

Rehder, D. (1992). Structure and function of vanadium compounds in living organisms. Biometals 5, 3-12.

Saponja, J. A. & Vogel, H. J. (1996). Metal-ion binding properties of the transferrins: a vanadium-51 NMR study. J Inorg Biochem 62, 253-270.

Schweizer, H. P. & Hoang, T. T. (1995). An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa. Gene 158, 15-22.

Schwyn, B. & Neilands, J. B. (1987). Universal chemical assay for the detection and determination of siderophores. Anal Biochem 160, 47-56.

Serino, L., Reimmann, C., Baur, H., Beyeler, M., Visca, P. & Haas, D. (1995). Structural genes for salicylate biosynthesis from chorismate in Pseudomonas aeruginosa. Mol Gen Genet 249, 217-228.

Serino, L., Reimmann, C., Visca, P., Beyeler, M., Della-Chiesa, V. & Haas, D. (1997). Biosynthesis of pyochelin and dihydroaeruginoic acid requires the iron-regulated pchDCBA operon in Pseudomonas aeruginosa. J Bacteriol 179, 248-257.

Simon, R., Priefer, U. & Puehller, A. (1983). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Bio/Technology 1, 784-791.

Srivastava, A. K. (2000). Anti-diabetic and toxic effects of vanadium compounds. Mol Cell Biochem 206, 177-182.

Stern, A., Davison, A. J., Wu, Q. & Moon, J. (1992). Desferrioxamine enhances the reactivity of vanadium (IV) toward ferri- and ferrocytochrome c. Free Radical Biol Med 12, 373-380.

Vasil, M. L. & Ochsner, U. A. (1999). The response of Pseudomonas aeruginosa to iron: genetics, biochemistry and virulence. Mol Microbiol 34, 399-413.

Visca, P., Colotti, G., Serino, L., Versili, D., Orsi, N. & Chiancone, E. (1992). Metal regulation of siderophore synthesis in Pseudomonas aeruginosa and functional effects of siderophore-metal complexes. Appl Environ Microbiol 58, 2886-2893.

Yanisch-Perron, C., Vieira, J. & Messing, J. (1985). Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33, 103-119.

Received 23 March 2000; revised 20 June 2000; accepted 29 June 2000.

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