title: Interaction of TonB with the outer membrane receptor FpvA of Pseudomonas aeruginosa.
abstract: Pyoverdine-mediated iron uptake by the FpvA receptor in the outer membrane of Pseudomonas aeruginosa is dependent on the inner membrane protein TonB1. This energy transducer couples the proton-electrochemical potential of the inner membrane to the transport event. To shed more light upon this process, a recombinant TonB1 protein lacking the N-terminal inner membrane anchor (TonB(pp)) was constructed. This protein was, after expression in Escherichia coli, purified from the soluble fraction of lysed cells by means of an N-terminal hexahistidine or glutathione S-transferase (GST) tag. Purified GST-TonB(pp) was able to capture detergent-solubilized FpvA, regardless of the presence of pyoverdine or pyoverdine-Fe. Targeting of the TonB1 fragment to the periplasm of P. aeruginosa inhibited the transport of ferric pyoverdine by FpvA in vivo, indicating an interference with endogenous TonB1, presumably caused by competition for binding sites at the transporter or by formation of nonfunctional TonB heterodimers. Surface plasmon resonance experiments demonstrated that the FpvA-TonB(pp) interactions have apparent affinities in the micromolar range. The binding of pyoverdine or ferric pyoverdine to FpvA did not modulate this affinity. Apparently, the presence of either iron or pyoverdine is not essential for the formation of the FpvA-TonB complex in vitro.
authors.affiliation: ESBS UMR7175, Récepteurs et Protéines Membranaires, Rue Sebastien Brant, BP 10413 F-67412 Illkirch Cedex, France.
authors.forename: Hendrik
authors.initials: H
authors.lastname: Adams
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Zeder-Lutz
authors.forename: Isabelle
authors.initials: I
authors.lastname: Schalk
authors.forename: Franc
authors.initials: F
authors.lastname: Pattus
authors.forename: Hervé
authors.initials: H
authors.lastname: Celia
mesh_terms: D001425:Bacterial Outer Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D007501:Iron / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D009842:Oligopeptides / Q000378:metabolism; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*
chemical_list: D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; C082326:FpvA protein, Pseudomonas aeruginosa; D008565:Membrane Proteins; D009842:Oligopeptides; C057616:tonB protein, Bacteria; C042453:pyoverdin; D007501:Iron