Pyoverdine (PvdI) is the major siderophore secreted by Pseudomonas aeruginosa PAOI in order to get access to iron. After being loaded with iron in the extracellular medium, PvdI is transported across the bacterial outer membrane by the transporter, FpvAI. We used the spectral properties of PvdI to show that in addition to Fe(3+), this siderophore also chelates, but with lower efficiencies, all the 16 metals used in our screening. Afterwards, FpvAI at the cell surface binds Ag(+), Al(3+), Cd(2+), Co(2+), Cu(2+), Fe(3+), Ga(3+), Hg(2+), Mn(2+), Ni(2+) or Zn(2+) in complex with PvdI. We used Inductively Coupled Plasma-Atomic Emission Spectrometry to monitor metal uptake in P. aeruginosa: TonB-dependent uptake, in the presence of PvdI, was only efficient for Fe(3+). Cu(2+), Ga(3+), Mn(2+) and Ni(2+) were also transported into the cell but with lower uptake rates. The presence of Al(3+), Cu(2+), Ga(3+), Mn(2+), Ni(2+) and Zn(2+) in the extracellular medium induced PvdI production in P. aeruginosa. All these data allow a better understanding of the behaviour of the PvdI uptake pathway in the presence of metals other than iron: FpvAI at the cell surface has broad metal specificity at the binding stage and it is highly selective for Fe(3+) only during the uptake process.

In order to get access to iron, Pseudomonas aeruginosa strain PAO1 produces two major siderophores pyoverdine (PVD) and pyochelin (PCH). Both siderophores are able to chelate many other metals in addition to iron. However, despite this property, only iron is transported efficiently into the bacteria by the PVD and PCH uptake pathways. Growth studies with P. aeruginosa strains showed a lower sensitivity to toxic metals for the siderophore-producing strain than for the mutants unable to produce siderophores. Moreover, addition of PVD or PCH to the growth medium of a siderophore-deficient strain considerably reduced the toxicity of toxic metals present at concentrations of 100 µM in iron-limited and iron-supplemented growth conditions. Measurement by Inductively Coupled Plasma-Atomic Emission Spectrometry of the concentration of metals present in bacteria incubated with metals in the presence or absence of PVD or PCH indicated that both siderophores were able to sequester metals from the extracellular medium of the bacteria, decreasing metal diffusion into the bacteria. Pyoverdine was able to sequester Al(3+) , Co(2+) , Cu(2+) , Eu(3+) , Ni(2+) , Pb(2+) , Tb(3+) and Zn(2+) from the extracellular medium, and PCH, Al(3+) , Co(2+) , Cu(2+) , Ni(2+) , Pb(2+) and Zn(2+) . Moreover, the presence of 100 µM Cu(2+) and Ni(2+) increased PVD production by 290% and 380%, respectively, in a medium supplemented with iron. All these data suggest that PVD and PCH may contribute to P. aeruginosa resistance to heavy metals.

To acquire iron, Pseudomonas aeruginosa secretes a major fluorescent siderophore, pyoverdine (PvdI), that chelates iron and shuttles it into the cells via the specific outer membrane transporter, FpvAI. We took advantage of the fluorescence properties of PvdI and its metal chelates as well as the efficient FRET between donor tryptophans in FpvAI and PvdI to follow the fate of the siderophore during iron uptake. Our findings with PvdI-Ga and PvdI-Cr uptake indicate that iron reduction is required for the dissociation of PvdI-Fe, that a ligand exchange for iron occurs, and that this dissociation occurs in the periplasm. We also observed a delay between PvdI-Fe dissociation and the rebinding of PvdI to FpvAI, underlining the kinetic independence of metal release and siderophore recycling. Meanwhile, PvdI is not modified but recycled to the medium, still competent for iron chelation and transport. Finally, in vivo fluorescence microscopy revealed patches of PvdI, suggesting that uptake occurs via macromolecular assemblies on the cell surface.Iron is an essential element for the growth of the vast majority of microorganisms. Under aerobic conditions, the abundance of free iron is limited by the very low solubility of ferric hydroxide. Thus, to maintain the required intracellular levels of iron, bacteria and fungi have developed efficient ferric ion-chelating agents, called siderophores (1), to scavenge iron from the extracellular environment and import it. A major siderophore produced by fluorescent Pseudomonas strains is pyoverdine forming a large class of mixed catecholate-hydroxamate siderophores characterized by a conserved dihydroxyquinolinederived chromophore to which a peptide chain of variable length and composition is attached (3, 4). In general, the uptake of ferric siderophores into Gram-negative bacteria involves a specific outer membrane transporter (OMT) and an inner membrane ABC transporter (5-7). The energy required for transport across the inner membrane is provided by ATP hydrolysis. The proton motive force of the inner membrane drives OMT-mediated transport across the outer membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (8, 9). The PvdI OMT (FpvAI) of Pseudomonas aeruginosa was cloned by Dean and Poole (10) in 1993, and its structure was recently solved (11). FpvAI (11), like FptA (12), FhuA (13, 14), FepA (15), and FecA (16, 17) (the outer membrane transporters of pyochelin in P. aeruginosa and of ferrichrome, ferric enterobactin, and ferric citrate in Escherichia coli, respectively) consists of a C-terminal ␤-barrel domain and an N-terminal plug domain filling the barrel. The binding site for the ferric siderophore is located above the plug, well outside the membrane, and is composed of residues of the plug and ␤-barrel domains. The binding site of FpvAI consists mostly of aromatic residues, including six Tyr residues and two Trp residues, and only three hydrophilic residues (11).The P. aeruginosa FpvAI is the best characterized pyoverdine OMT, whereas only three o...

a b s t r a c tPyoverdine I (PVDI) is the major siderophore produced by Pseudomonas aeruginosa to import iron. Biosynthesis of this chelator involves non-ribosomal peptide synthetases and other enzymes. PvdQ is a periplasmic enzyme from the NTN hydrolase family and is involved in the final steps of PVDI biosynthesis. A pvdQ mutant produces two non-fluorescent PVDI precursors with a higher molecular mass than PVDI. In the present study, we describe the use of mass spectrometry to determine the structure of these PVDI precursors and show that they both contain a unformed chromophore like ferribactin, and either a myristic or myristoleic chain that must be removed before PVDI is secreted into the extracellular medium.