The PvdRT-OpmQ efflux pump controls the metal selectivity of the iron uptake pathway mediated by the siderophore pyoverdine in Pseudomonas aeruginosa Mélissa Hannauer, 1 Armelle Braud, 1 Françoise Hoegy, 1 Pascale Ronot, 2 Anne Boos 2 and Isabelle J. Schalk 1 * 1 UMR7242, Université de Strasbourg-CNRS, ESBS, Blvd Sébastien Brant, F-67413 Illkirch, Strasbourg, France. 2 Institut Pluridisciplinaire Hubert Curien, UMR 7178, CNRS-ECPM-Université de Strasbourg, 25 rue Becquerel, 67087 Strasbourg Cedex, France. Summary Pyoverdine (PVD) is the major siderophore produced by Pseudomonas aeruginosa for iron acquisition. PvdRT-OpmQ is an ATP-dependent efflux pump involved in the secretion of newly synthesized pyoverdine (PVD) and of PVD that has transported and released its iron into the bacterium from the peri- plasm into the extracellular medium. This iron uptake pathway also involves an outer membrane trans- porter, FpvA, for PVD–Fe uptake from the extracellu- lar medium into the periplasm. In binding assays, FpvA bound PVD in complex with many different metals, with affinities from 2.9 nM for PVD–Fe to 13 mM for PVD–Al. Uptake assays with various FpvA and PvdRT-OpmQ mutants, monitored by inductively coupled plasma-atomic emission spectrometry (ICP- AES) for metal detection, and by fluorescence for PVD detection, showed that both metals and PVD accumu- lated in P. aeruginosa, due to the uptake of these compounds via the FpvA/PVD pathway. Higher levels of accumulation were observed in the absence of PvdRT-OpmQ expression. Thus, FpvA has a broad metal specificity for both the binding and uptake of PVD–metal complexes, and the PvdRT-OpmQ efflux pump exports unwanted metals complexed with PVD from the bacterium. This study provides the first evi- dence of efflux pump involvement in the export of unwanted siderophore–metal complexes and insight into the molecular mechanisms involved controlling the metal selectivity of siderophore-mediated iron uptake pathways. Introduction To get access to iron, bacteria produce and release in their environment siderophores (Chu et al., 2010; Hider and Kong, 2010), which are small molecules (MW between 200 and 2000 Da) characterized by an extremely high affinity for iron. More than 500 different siderophore structures have been described to date. Microorganisms in the rhizosphere produce siderophores at concentra- tions that have been estimated somewhere between the micromolar and the millimolar range. Bacteria also produce these compounds during infection, to allow them to acquire iron from the host; siderophores are therefore considered to be virulence factors (Takase et al., 2000; Lamont et al., 2009; Taguchi et al., 2010). Once the iron in the extracellular medium has been chelated, the resulting ferrisiderophore complex is transported into the bacterium via specific membrane transporters. Pyoverdine (PVD) is the principal siderophore produced by the opportunist pathogen Pseudomonas aeruginosa (Schalk, 2008). PVD is a partly cyclic octapeptide linked to a chromophore derived from 2,3-diamino-6,7-dihydroxyquinoline, which renders the siderophore both coloured and fluorescent (Demange et al., 1990; Wasielewski et al., 2002). PVD synthesis begins in the bacterial cytoplasm, with the assembly of a non-fluorescent precursor by non- ribosomic peptide synthetases (NRPSs) (Visca et al., 2007), and ends in the periplasm with cyclization of the chromophore moiety (Yeterian et al., 2010a). The non- fluorescent precursor is transported across the inner membrane into the periplasm, probably by the ABC trans- porter PvdE (Yeterian et al., 2010a), and the PVD subse- quently produced is transported across the outer membrane and released into the extracellular medium by the ATP-dependent PvdRT-OpmQ efflux system (Han- nauer et al., 2010). This efflux pump thought to display an organization similar to that of other bacterial efflux pumps (Li and Nikaido, 2009; Nikaido and Takatsuka, 2009), with PvdT predicted to be an inner membrane protein, PvdR a periplasmic adaptor protein and OpmQ an outer mem- brane protein with a porin-like b-barrel and a large Received 26 May, 2011; revised 19 October, 2011; accepted 31 October, 2011. *For correspondence. E-mail isabelle.schalk@ unistra.fr; Tel. (+33) 3 68 85 47 19; Fax (+33) 3 68 85 48 29. Environmental Microbiology (2012) 14(7), 1696–1708 doi:10.1111/j.1462-2920.2011.02674.x © 2011 Society for Applied Microbiology and Blackwell Publishing Ltd