Pyoverdine-Mediated Iron Uptake in Pseudomonas aeruginosa : the Tat System Is Required for PvdN but Not for FpvA Transport

Abstract

Under iron-limiting conditions, Pseudomonas aeruginosa PAO1 secretes a fluorescent siderophore called pyoverdine (Pvd). After chelating iron, this ferric siderophore is transported back into the cells via the outer membrane receptor FpvA. The Pvd-dependent iron uptake pathway requires several essential genes involved in both the synthesis of Pvd and the uptake of ferric Pvd inside the cell. A previous study describing the global phenotype of a tat-deficient P. aeruginosa strain showed that the defect in Pvd-mediated iron uptake was due to the Tat . Using biochemical and biophysical tools, we showed that despite its predicted Tat signal sequence, FpvA is correctly located in the outer membrane of a tat mutant and is fully functional for all steps of the iron uptake process (ferric Pvd uptake and recycling of Pvd on FpvA after iron release). However, in the tat mutant, no Pvd was produced. This suggested that a key element in the Pvd biogenesis pathway must be exported to the periplasm by the Tat pathway. We located PvdN, a still unknown but essential component in Pvd biogenesis, at the periplasmic side of the cytoplasmic membrane and showed that its export is Tat dependent. Our results further support the idea that a critical step of the Pvd biogenesis pathway involving PvdN occurs at the periplasmic side of the cytoplasmic membrane.

PvdN Enzyme Catalyzes a Periplasmic Pyoverdine Modification

“…As expected, the ⌬pvdP control strain could not produce fluorescent pyoverdine and did not grow on EDDHA-containing medium. However, unlike what was expected from the earlier interposon mutagenesis studies that could not exclude polar effects in the operon (6,10,11), the scar-less ⌬pvdN mutant strain was not impaired in pyoverdine formation, nor did the deletion affect growth on iron-depleted medium. In fact, the ⌬pvdN strain showed the same phenotype as the WT strain on both media, producing approximately equivalent (24), showing the fluorophore and the position of the modified N-terminal residue (R 1 ), which are the regions where periplasmic maturation occurs.…”

“…While significant progress has been made with PvdQ and PvdP (5,8,26), the function of any of the proteins encoded by the pvdMNO operon has remained unclear. As interposon mutagenesis suggested essential roles of these proteins for pyoverdine production, it was seemingly not possible to address the individual functions of these enzymes (6,10,11). Interestingly, we realized that an in-frame deletion of the pvdN gene caused a much milder phenotype than the interposon mutation, suggesting that polar effects might disturb the interpretation of the former approaches, which was already considered by the authors of the original deletion study (10).…”

“…PvdN is known to be a Tat-dependently translocated protein (11), and the crystal structure of P. aeruginosa PvdN demonstrated an incorporated PLP cofactor (12). In Pseudomonas taetrolens, it has been demonstrated that PLP could be incorporated into a Sec-dependently translocated periplasmic amino acid racemase after translocation (27), and no PLP insertion into Tat-dependently translocated enzymes has been described so far.…”

Iron Gate: the Translocation System

“…However, this new prediction program does not solve all problems, because it still predicts that other TonBdependent transducers, such as FoxA and Pseudomonas putida PupB (8), do have a potential Tat signal sequence. Based on the work of Voulhoux et al (15) and knowledge of the general structure of TonB-dependent receptors, it is not likely that these receptors are translocated via Tat; this finding shows the importance of experimental analyses of these predictions. Voulhoux et al also show that, although FpvA translocation is not Tat dependent, the production of pyoverdine is, as was also described previously (10,15).…”

“…Based on the work of Voulhoux et al (15) and knowledge of the general structure of TonB-dependent receptors, it is not likely that these receptors are translocated via Tat; this finding shows the importance of experimental analyses of these predictions. Voulhoux et al also show that, although FpvA translocation is not Tat dependent, the production of pyoverdine is, as was also described previously (10,15). The authors convincingly demonstrate that at least one of the proteins involved in the biogenesis of pyoverdine, PvdN, a still unknown but essential component in pyoverdine biogenesis, is transported into the periplasm by the Tat pathway.…”