The Ferrichrome Uptake Pathway in Pseudomonas aeruginosa Involves an Iron Release Mechanism with Acylation of the Siderophore and Recycling of the Modified Desferrichrome

The uptake of iron into Pseudomonas aeruginosa is mediated by two major siderophores produced by the bacterium, pyoverdine and pyochelin. The bacterium is also able of utilize several heterologous siderophores of bacterial or fungal origin. In this work, we have investigated the iron uptake in P. aeruginosa PAO1 by the heterologous ferrichrome siderophore. ⁵⁵Fe uptake assays showed that ferrichrome is transported across the outer membrane primarily (80%) by the FiuA receptor and to a lesser extent (20%) by a secondary transporter. Moreover, we demonstrate that like in the uptake of ferripyoverdine and ferripyochelin, the energy required for both pathways of ferrichrome uptake is provided by the inner membrane protein TonB1. Desferrichrome-⁵⁵Fe uptake in P. aeruginosa was also dependent on the expression of the permease FiuB, suggesting that this protein is the inner membrane transporter of the ferrisiderophore. A biomimetic fluorescent analogue of ferrichrome, RL1194, was used in vivo to monitor the kinetics of iron release from ferrichrome in P. aeruginosa in real time. This dissociation involves acylation of ferrichrome and its biomimetic analogue RL1194 and recycling of both modified siderophores into the extracellular medium. FiuC, an N-acetyltransferase, is certainly involved in this mechanism of iron release, since its mutation abolished desferrichrome-⁵⁵Fe uptake. The acetylated derivative reacts with iron in the extracellular medium and is able to be taken up again by the cells. All these observations are discussed in light of the current knowledge concerning ferrichrome uptake in P. aeruginosa and in Escherichia coli.Iron is essential for life for practically all living organisms and plays a number of key roles in biology. DNA and RNA synthesis, glycolysis, energy generation by electron transport, nitrogen fixation, and photosynthesis are examples of processes in which iron-containing enzymes play vital roles. However, under physiological conditions iron forms highly insoluble ferric hydroxide complexes, which severely limits its bioavailability. To overcome the problem of iron inaccessibility, bacteria excrete high-affinity iron chelators termed siderophores, which are able to solubilize iron and deliver it into the cells (3, 64).Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is capable of infecting a wide variety of animal, insects, and plants. As a human pathogen, P. aeruginosa is the leading source of Gram-negative nosocomial infections (59) and causes chronic lung infections in approximately 90% of individuals suffering from cystic fibrosis (40). Under iron-limited conditions, P. aeruginosa produces two major siderophores, pyoverdine (PVD) (62) and pyochelin (PCH) (15). P. aeruginosa is also capable of utilizing numerous siderophores secreted by other microorganisms: pyoverdins from other pseudomonas, enterobactin (49), cepabactin (45), mycobactin and carboxymycobactin (38), fungal siderophores (ferrichrome 39]; deferrioxamines 39, 60]; and desferrichrysin, desferricrocin, and coprogen 44]), and natural occurring chelators such as citrate (14, 23) (for a review, see reference 47).In Gram-negative bacteria, the uptake of ferrisiderophores always involves a specific transporter at the level of the outer membrane (4). The energy required for this process is provided by the proton motive force (PMF) of the inner membrane by means of an inner membrane complex comprising TonB, ExbB, and ExbD (21, 51, 63). In silico analysis of the P. aeruginosa genome (http://www.pseudomonas.com) revealed 32 genes encoding putative TonB-dependent transporters (13), of which only 12 are involved in metal (mostly iron) uptake (38). FpvA and FpvB are the outer membrane transporters involved in the uptake of PVD-Fe (19, 48), and FptA transports PCH-Fe (25). Concerning the heterologous siderophores, there are two transporters, FoxA and FiuA, involved in the transport of ferrioxamine B and ferrichrome (39). The mechanism involved in the translocation of ferrisiderophores across the outer membrane by the TonB-dependent transporters has been studied mostly in E. coli (for a review, see reference 5) and in the case of P. aeruginosa has been studied only for the FpvA/PVD and the FptA/PCH systems. The structures of FpvA (8, 11, 65) and FptA (12) have been solved and their interactions with PVD and PCH investigated at the molecular level (26, 27, 45, 53). Three tonB genes, encoding the energy coupler TonB, have been found in the P. aeruginosa genome, i.e., tonB1, tonB2, and tonB3. Disruption of tonB1 abrogates PVD- and PCH-mediated iron uptake (50, 58) and heme uptake (67). Inactivation of tonB2 has no adverse effect on iron or heme acquisition, but tonB1 tonB2 double mutants are more compromised with respect to growth in iron-restricted medium than is a single tonB1 knockout mutant (67). Mutation of tonB3 appears to result in defective twitching motility (28), and the gene product is most likely not involved in iron uptake.In P. aeruginosa, many ferrisiderophore outer membrane transporters are also involved in a signaling cascade regulating the expression of genes involved in iron uptake. This is the case for FpvA (PVD uptake), FoxA (ferrioxamine), and FiuA (ferrichrome) (38, 39, 43, 61). Such a signaling cascade involves an extracytoplasmic function (ECF) sigma factor and an inner membrane anti-sigma factor. Equivalent cell surface signaling is present in Escherichia coli for ferricitrate uptake by FecA but not for ferrichrome, ferrioxamine, and enterobactin uptake by FhuA, FhuE, and FepA, respectively.Little is known about the translocation of ferrisiderophores across the inner membrane in P. aeruginosa. In E. coli, this step involves a specific ABC transporter for almost every siderophore used by this bacterium: FhuBCD for the uptake of ferrichrome and ferrioxamine (33-36), FecBCD for the uptake of ferricitrate (6, 56) and, FepBCDG for the uptake of ferrienterobactin (9). In P. aeruginosa, the only characterized inner membrane siderophore transport protein is FptX, a proton motive force-dependent permease, which functions in PCH-Fe utilization (16). The inner membrane FoxB is involved in the utilization of ferrichrome and ferrioxamine B, but it remains to be determined whether this protein functions in the transport of ferrisiderophore or in the release of iron from ferrichrome or ferrioxamine (17). The genome clearly shows a fepBCDG homologue for the transport of ferrienterobactin. For the other iron uptake pathways present in P. aeruginosa, the transporters involved at the level of the inner membrane have not been identified. An import ABC transporter is present in the pvd locus (PA2407 to PA2410; http://www.pseudomonas.com), but its mutation does not affect PVD-Fe uptake (46).In P. aeruginosa, the mechanism of ferrisiderophore dissociation has been investigated only for the PVD pathway. This step occurs in the periplasm by a mechanism involving no chemical siderophore modification but involving a reduction of iron and a recycling of the siderophore into the extracellular medium by the PvdRT-OpmQ efflux pump (20, 54, 66). In E. coli, the mechanism of ferrisiderophore dissociation has been investigated for the ferrichrome and ferrienterobactin pathways. Iron release from ferrichrome occurs in the cytoplasm and probably involves iron reduction (41) followed by acetylation of the siderophore and its recycling into the growth medium (24). For the ferrienterobactin pathway, a cytoplasmic esterase hydrolyzes the siderophore (7).In the present work, we have investigated the ferrichrome pathway in P. aeruginosa using both ferrichrome and a fluorescently labeled biomimetic ferrichrome analogue. We evaluated the siderophore properties of the fluorescent analogue and identified the different transporters involved in the uptake across the outer and inner membranes. Furthermore, we demonstrated that following ferrichrome uptake, iron is released from the siderophore by a mechanism involving an acetylation of the chelator and the modified desferrichrome is secreted back into the growth medium.