Copurification of the FpvA Ferric Pyoverdin Receptor ofPseudomonasaeruginosawith Its Iron-Free Ligand:  Implications for Siderophore-Mediated Iron Transport

Abstract

The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.

The Metal Dependence of Pyoverdine Interactions with Its Outer Membrane Receptor FpvA

“…The purification of an iron-free FpvA-Pvd complex from the Pvd-producing and FpvA-overexpressing strain K691(pPVR2) has been previously reported (26) and has been used to solve the crystal structure of the FpvA-Pvd complex (6). Although those authors noted in their description of the structure that there was a significant density in the Pvd molecule where an iron would normally be bound, it was not attributed to a metal and thus was not modeled.…”

“…The TonB-dependent OMTs include the receptors for a wide variety of siderophores as well as for other, non-iron-containing molecules, yet to date, they have all been found to have a conserved structure with a similar binding site for the transported molecule (30). However, one intriguing difference among the siderophore receptors was the reported ability of the pyoverdine (Pvd) receptor from Pseudomonas aeruginosa (FpvA) (26) and the ferric citrate receptor from Escherichia coli (FecA) (32) to bind their siderophores in the absence of Fe. More recently, iron-free siderophore binding was reported for FptA and FhuA, the P. aeruginosa pyochelin and E. coli ferrichrome receptors, respectively (15), and the reported affinities of the iron-free siderophores are always 5-to 20-fold lower than those of the ferric siderophores, which have dissociation constants in the range of 1 nM.…”

Siderophores for molecular imaging applications

“…A number of investigations were made with 3 H, 14 C, 55 Fe and 59 Fe labelled siderophores mainly to study iron transport or siderophore uptake mechanisms in microorganisms or plants [e.g. [42][43][44] unsuitable for molecular imaging and, therefore, cannot be used for detection of microbial infections in vivo. By contrast, radionuclides used in the studies of In-have found widespread use in nuclear medicine for SPECT imaging.…”

FpvA-Mediated Ferric Pyoverdine Uptake in Pseudomonas aeruginosa: Identification of Aromatic Residues in FpvA Implicated in Ferric Pyoverdine Binding and Transport

“…1B), they were not deemed sufficiently close to be implicated in pyoverdine binding (6). Still, it is interesting to note that W362 and W391, identified here as important for ferric pyoverdine binding, were implicated (6) as two of three tryptophan residues of FpvA responsible for fluorescence energy transfer (FRET) with the pyoverdine chromophore in earlier studies of FpvA-pyoverdine binding (25,27). Moreover, these tryptophans also appear (6) to contribute to FRET in in vitro-reconstituted FpvA complexed with metal (i.e.…”

“…A second receptor for type I pyoverdine, FpvB, has also recently been reported for P. aeruginosa (10). The FpvA receptor, like other ferric siderophore receptors (12), has been shown to bind both iron-free and iron-bound siderophores (25)(26)(27), although there appear to be differences in the ways that iron-free and iron-bound pyoverdines interact with FpvA (5, 9). Still, both compete with a common or at least overlapping site on FpvA (5), and iron-bound pyoverdine effectively displaces iron-free pyoverdine on the receptor during transport (24,25).…”