title: Mechanism of ferripyoverdine uptake by Pseudomonas aeruginosa outer membrane transporter FpvA: no diffusion channel formed at any time during ferrisiderophore uptake.
abstract: To get access to iron, Pseudomonas aeruginosa produces the siderophore pyoverdine (PVD), composed of a fluorescent chromophore linked to an octapeptide, and its corresponding outer membrane transporter FpvA. This transporter is composed of three domains: a β-barrel inserted into the membrane, a plug that closes the channel formed by the barrel, and a signaling domain in the periplasm. The plug and the signaling domain are separated by a sequence of five residues called the TonB box, which is necessary for the interaction of FpvA with the inner membrane TonB protein. Genetic deletion of the plug domain resulted in the presence of a β-barrel in the outer membrane unable to bind and transport PVD-Fe. Expression of the soluble plug domain with the TonB box inhibited PVD-(55)Fe uptake most likely through interaction with TonB in the periplasm. A reconstituted FpvA in the bacterial outer membrane was obtained by the coexpression of separately encoded plug and β-barrel domains, each endowed with a signal sequence and a signaling domain. This resulted in polypeptide complementation after secretion across the cytoplasmic membrane. The reconstituted FpvA bound PVD-Fe with the same affinity as wild-type FpvA, indicating that the resulting transporter is correctly folded and reconstituted in the outer membrane. PVD-Fe uptake was TonB-dependent but 75% less efficient compared to wild-type FpvA. These data are consistent with a gated mechanism in which no open channel with a complete removal of the plug domain for PVD-Fe diffusion is formed in FpvA at any point during the uptake cycle.
authors.affiliation: UMR7242, Université de Strasbourg-CNRS, ESBS, Blvd Sébastien Brandt, F-67513 Illkirch, France.
authors.forename: Mirella
authors.initials: M
authors.lastname: Nader
authors.forename: Laure
authors.initials: L
authors.lastname: Journet
authors.forename: Ahmed
authors.initials: A
authors.lastname: Meksem
authors.forename: Laurent
authors.initials: L
authors.lastname: Guillon
authors.forename: Isabelle J
authors.initials: IJ
authors.lastname: Schalk
mesh_terms: D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D001692:Biological Transport; D047468:Immunoprecipitation; D008024:Ligands; D008565:Membrane Proteins / Q000378:metabolism; D050505:Mutant Proteins / Q000378:metabolism; D009842:Oligopeptides / Q000493:pharmacokinetics*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D021382:Protein Sorting Signals; D011550:Pseudomonas aeruginosa / Q000378:metabolism*; D011993:Recombinant Fusion Proteins; D017262:Siderophores / Q000493:pharmacokinetics*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C082326:FpvA protein, Pseudomonas aeruginosa; D008024:Ligands; D008565:Membrane Proteins; D050505:Mutant Proteins; D009842:Oligopeptides; D010446:Peptide Fragments; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D017262:Siderophores; C057616:tonB protein, Bacteria; C042453:pyoverdin