SummaryUnder iron limitation, Pseudomonas aeruginosa secretes a fluorescent siderophore called pyoverdin, which, after complexing iron, is transported back into the cell via its outer membrane receptor FpvA. Previous studies demonstrated co-purification of FpvA with iron-free PaA and reported similar binding affinities of iron-free pyoverdin and ferric-pyoverdin to purified FpvA. The fluorescence resonance energy transfer between iron-free PaA and the FpvA receptor here reveals the existence of an FpvA±pyoverdin complex in P. aeruginosa in vivo, suggesting that the pyoverdin-loaded FpvA is the normal state of the receptor in the absence of iron. Using tritiated ferricpyoverdin, it is shown that iron-free PaA binds to the outer membrane but is not taken up into the cell, and that in vitro and, presumably, in vivo ferric-pyoverdin displaces the bound iron-free pyoverdin on FpvA± PaA to form FpvA±PaA-Fe complexes. In vivo, the kinetics of formation of this FpvA±PaA-Fe complex are more than two orders of magnitude faster than in vitro and depend on the presence of TonB. In P. aeruginosa, two tonB genes have been identified (tonB1 and tonB2). TonB1 is directly involved in ferric-pyoverdin uptake, and TonB2 seems to be able partially to replace TonB1 in its role in iron acquisition. However, no effect of TonB1 or TonB2 on the apparent affinity of free pyoverdin to FpvA was observed, and a 17-fold difference was measured between the affinities of the two forms of pyoverdin (PaA and PaA-Fe) to FpvA in the absence of TonB1 or TonB2. The mechanism of iron uptake in P. aeruginosa via the pyoverdin pathway is discussed in view of these new findings.

Pseudomonas aeruginosa is particularly resistant to most all the antibiotics presently available, essentially because of the very low permeability of its outer membrane. To overcome this, we synthesized four siderophore-based antibiotics formed by two quinolones - norfloxacin and benzonaphthyridone - bound to the pyoverdin of P. aeruginosa ATCC 15692 via two types of spacer arms: one stable and the other readily hydrolyzable. From the comparison of their antibacterial properties with those of the two unbound quinolones, we reached the following conclusions: (a) The adducts inhibit Escherichia coli's gyrase showing that the dissociation of the compounds is not necessary for their activity. However, the presence of the pyoverdin moiety on the molecule decreases the inhibition activity compared to the antibiotic alone. (b) They facilitate the uptake of (55)Fe using the specific pyoverdin-mediated iron-transport system of the bacterium. No uptake was observed either with P. aeruginosa ATCC 27853, which produces a structurally different pyoverdin, or with P. aeruginosa K690, which is a mutant of P. aeruginosa ATCC 15692 lacking FpvA, the outer-membrane pyoverdin receptor. (c) MIC determinations have shown that only strains P. aeruginosa ATCC 15692 and the derived outer-membrane receptor-producing but pyoverdin-deficient P. aeruginosa IA1 mutant present higher susceptibility to the pyoverdin-quinolone adducts, whereas P. aeruginosa ATCC 27853 and K690 are much more resistant. (d) Growth inhibition by these adducts confirmed these results and showed that the adducts with the hydrolyzable spacer arm have better activity than those with the stable one and that the labile spacer arm adducts present much higher activity than the quinolones alone. These results show clearly that the penetration of the antibiotic into the cells is favored when this latter is coupled with pyoverdin: Only the strains possessing the appropriate outer-membrane receptor present higher susceptibility to the adduct. In this case the antibiotic uses the pyoverdin-mediated iron-transport system. Furthermore, better efficiency is obtained when the spacer arm is labile and favors the antibiotic release inside the cell, allowing better inhibition of gyrase.

Azotobacter vinelandii, a nitrogen-fixing soil bacterium, secretes in iron deficiency azotobactin delta, a highly fluorescent pyoverdin-like chromopeptidic hexadentate siderophore. The chromophore, derived from 2,3-diamino-6,7 dihydroxyquinoline, is bound to a peptide chain of 10 amino acids: (L)-Asp-(D)-Ser-(L)-Hse-Gly-(D)-beta-threo-HOAsp-(L)-Ser-(D)-Cit-(L)-Hse-(L)-Hse lactone-(D)-N(delta)-Acetyl, N(delta)-HOOrn. Azotobactin delta has three different iron(III) binding sites which are one hydroxamate group at the C-terminal end of the peptidic chain (N(delta)-Acetyl, N(delta)-HOOrn), one alpha-hydroxycarboxylic function in the middle of the chain (beta-threo-hydroxyaspartic acid), and one catechol group on the chromophore. The coordination properties of its iron(III) and iron(II) complexes were measured by spectrophotometry, potentiometry, and voltammetry after the determination of the acid-base functions of the uncomplexed free siderophore. Strongly negatively charged ferric species were observed at neutral p[H]'s corresponding to a predominant absolute configuration Lambda of the ferric complex in solution as deduced from CD measurements. The presence of an alpha-hydroxycarboxylic chelating group does not decrease the stability of the iron(III) complex when compared to the main trishydroxamate siderophores or to pyoverdins. The value of the redox potential of ferric azotobactin is highly consistent with a reductive step by physiological reductants for the iron release. Formation and dissociation kinetics of the azotobactin delta ferric complex point out that both ends of this long siderophore chain get coordinated to Fe(III) before the middle. The most striking result provided by fluorescence measurements is the lasting quenching of the fluorophore in the course of the protonation of the ferric azotobactin delta complex. Despite the release of the hydroxyacid and of the catechol, the fluorescence remains indeed quenched, when iron(III) is bound only to the hydroxamic acid, suggesting a folded conformation at this stage, around the metal ion, in contrast to the unfolded species observed for other siderophores such as ferrioxamine or pyoverdin PaA.