The siderophore pyoverdine (PVD) is a primary virulence factor of the human pathogenic bacterium Pseudomonas aeruginosa, acting as both an iron carrier and a virulence-related signal molecule. By exploring a number of P. aeruginosa candidate systems for PVD secretion, we identified a tripartite ATP-binding cassette efflux transporter, here named PvdRT-OpmQ, which translocates PVD from the periplasmic space to the extracellular milieu. We show this system to be responsible for recycling of PVD upon internalization by the cognate outer-membrane receptor FpvA, thus making PVD virtually available for new cycles of iron uptake. Our data exclude the involvement of PvdRT-OpmQ in secretion of de novo synthesized PVD, indicating alternative pathways for PVD export and recycling. The PvdRT-OpmQ transporter is one of the few secretion systems for which substrate recognition and extrusion occur in the periplasm. Homologs of the PvdRT-OpmQ system are present in genomes of all fluorescent pseudomonads sequenced so far, suggesting that PVD recycling represents a general energysaving strategy adopted by natural Pseudomonas populations.ATP-binding-cassette transporter ͉ periplasm ͉ iron ͉ fluorescent Pseudomonas ͉ secretion

SummaryIn Pseudomonas aeruginosa the iron starvation sigma factor PvdS directs the transcription of pyoverdine and virulence genes under iron limitation. PvdS activity is modulated by pyoverdine through the surface signalling cascade involving the FpvA receptor and the inner membrane-spanning sensor FpvR. To gain insight into the molecular mechanisms enabling PvdS to compete with the major sigma RpoD for RNA polymerase (RNAP) binding, we determined the intracellular levels of RNAP, RpoD and PvdS in P. aeruginosa PAO1, and the effect of pyoverdine signalling on PvdS activity. Under iron limitation, P. aeruginosa contains 2221 and 933 molecules of RNAP and RpoD per cell respectively. PvdS attains 62% of RpoD levels. The high PvdS content is partly offset by retention of 30% of PvdS on the membrane, lowering the concentration of cytosolic PvdS to 45% of RpoD levels. RNAP purification from iron-starved P. aeruginosa cells demonstrated that PvdS-RNAP is poorly represented compared with RpoD-RNAP (1 and 27% of total RNAP respectively). Pyoverdine signalling does not affect the PvdS cellular content but facilitates PvdS release from the membrane, increasing its cytosolic concentration from 35% in both pvdF and fpvA signalling mutants to 70% in the wild type and 83% in the fpvR mutant.

The Gram-negative bacterium Pseudomonas aeruginosa is a main cause of infection in hospitalized, burned, immunocompromised, and cystic fibrosis patients. Many processes essential for P. aeruginosa pathogenesis, e.g., nutrient uptake, antibiotic resistance, and virulence, take place in the cell envelope and depend on components residing in the periplasmic space. Recent high-throughput studies focused on P. aeruginosa membrane compartments. However, the composition and dynamics of its periplasm remain largely uncharacterized. Here, we report a detailed description of the periplasmic proteome of the wild-type P. aeruginosa strain PAO1 by 2-DE and MALDI-TOF/TOF analysis. Three extraction methods were compared at proteome level in order to achieve the most reliable and comprehensive periplasmic protein map. A total of 495 spots representing 395 different proteins were identified. Most of the high intensity spots corresponded to periplasmic proteins, while cytoplasmic contaminants were mainly detected among faint spots. The majority of the identified periplasmic proteins is involved in transport, cell-envelope integrity, and protein folding control. Notably, more than 30% still has an unpredicted function. This work provides the first overview of the P. aeruginosa periplasm and offers the basis for future studies on periplasmic proteome changes occurring during P. aeruginosa adaptation to different environments and/or antibiotic treatments.