Françoise Hoegy

Iron is an important element for almost all forms of life. In order to get access to this essential nutriment, Pseudomonads produce two major siderophores, pyoverdine PVD and pyochelin (PCH). Uptake of iron in bacterial cells can be monitored accurately using (55)Fe. Bacteria cells are incubated in the presence of either PVD or PCH loaded with (55)Fe. After incubation, extracellular iron ions are separated from those accumulated in the bacteria cells by either centrifugation or filtration on glass microfiber filters, for the PCH and PVD assays, respectively. (55)Fe contained in the harvested cells on the filter or in the cell pellet is counted in scintillation cocktail. The number of moles of (55)Fe transported can be determined using the specific activity of the radionuclide.

Siderophores are small organic chelators (of molecular weight between 200 and 2,000 Da), having a very high affinity for iron (10(17)-10(43) M(-1)). They are synthesized by bacteria and secreted into their environment in order to get access to iron, an essential element for bacterial growth. Pyoverdine (also called fluorescins or pseudobactins) and pyochelin are the two major siderophores produced by Pseudomonas aeruginosa in iron-limited media. Methods to specifically detect and measure the amount of pyoverdine and pyochelin in a bacterial culture are provided here. These methods are based on the spectral properties of these two siderophores.

TonB-dependent iron transporters present in the outer membranes of Gram-negative bacteria transport ferric-siderophore complexes into the periplasm. This requires proton motive force and an integral inner membrane complex, TonB-ExbB-ExbD. Recognition of iron-free siderophores by TonB-dependent outer membrane transporters (OMT) has only been described for a subfamily called OMT(N). These OMT(N)s have an additional domain at the N terminus, which interacts with an inner membrane regulatory protein to activate a cytoplasmic sigma factor. This induces transcription of iron transport genes. Here we showed that the ability to bind aposiderophores is not specific to the OMT(N) subfamily but may be a more general feature of OMTs. FhuA, the ferrichrome OMT in Escherichia coli, and FptA, the pyochelin (Pch) OMT in Pseudomonas aeruginosa, were both able to bind in vitro and in vivo the apo-forms and the ferric forms of their corresponding siderophore at a common binding site. FptA produced in P. aeruginosa cells grown in an iron-deficient medium copurifies with a ligand that, as characterized by fluorescence, is iron-free Pch. As described previously for the FpvA transporter (pyoverdine OMT in P. aeruginosa), it appears that in conditions of iron limitation all the FptA receptors at the cell surface are loaded with apoPch. This FptA-Pch complex is less stable in vitro than the previously described copurified FpvA-Pvd complex and can be loaded with iron in vitro in the presence of Pch-Fe, citrate-Fe, or ferrichrome-Fe. These findings improved our understanding of the iron uptake mechanism via siderophores in Gram-negative bacteria.

Pyochelin (Pch) and enantio-pyochelin (EPch) are enantiomer siderophores that are produced by Pseudomonas aeruginosa and Pseudomonas fluorescens, respectively, under iron limitation. Pch promotes growth of P. aeruginosa when iron is scarce, and EPch carries out the same biological function in P. fluorescens. However, the two siderophores are unable to promote growth in the heterologous species, indicating that siderophore-mediated iron uptake is highly stereospecific. In the present work, using binding and iron uptake assays, we found that FptA, the Fe-Pch outer membrane transporter of P. aeruginosa, recognized (K(d) = 2.5 +/- 1.1 nm) and transported Fe-Pch but did not interact with Fe-EPch. Likewise, FetA, the Fe-EPch receptor of P. fluorescens, was specific for Fe-EPch (K(d) = 3.7 +/- 2.1 nm) but did not bind and transport Fe-Pch. Growth promotion experiments performed under iron-limiting conditions confirmed that FptA and FetA are highly specific for Pch and EPch, respectively. When fptA and fetA along with adjacent transport genes involved in siderophore uptake were swapped between the two bacterial species, P. aeruginosa became able to utilize Fe-EPch as an iron source, and P. fluorescens was able to grow with Fe-Pch. Docking experiments using the FptA structure and binding assays showed that the stereospecificity of Pch recognition by FptA was mostly due to the configuration of the siderophore chiral centers C4'' and C2'' and was only weakly dependent on the configuration of the C4' carbon atom. Together, these findings increase our understanding of the stereospecific interaction between Pch and its outer membrane receptor FptA.

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.

Previously it has been postulated that the pyochelin-Fe outer membrane transporter, FptA, is involved in the uptake of catechol-substituted cephalosporins in Pseudomonas aeruginosa. Iron uptake and antibacterial activity studies on different mutants showed clearly that FptA is unable to bind and transport these antibiotics.