Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa.
FpvA is an outer membrane transporter involved in iron uptake by the siderophore pyoverdine (Pvd) in Pseudomonas aeruginosa. This transporter, like all other proteins of the same family, consists of a transmembrane 22 beta-stranded barrel occluded by a plug domain. The beta-strands of the barrel are connected by large extracellular loops and short periplasmic turns. Site-directed mutagenesis was carried out on FpvA to identify the extracellular loops or parts of these loops involved in the various stages of Pvd-Fe uptake. The G286C, W362C, and W434C mutations in loops L1, L3, and L4, respectively, disturbed the binding of the apo siderophore, as shown by time-resolved fluorescence spectroscopy. Iron uptake experiments followed by fluorescence resonance energy transfer (FRET) or using 55Fe indicated that residues W434 and G701 and, therefore, loops L4 and L9 must be involved in Pvd-Fe uptake by FpvA. The two corresponding mutants incorporated smaller than normal amounts of 55Fe into cells, and no Pvd recycling on FpvA was observed after iron release. Surprisingly, the S603C mutation in loop L7 increased the amount of Pvd-Fe transported. Our results suggest that W434 (L4), S603 (L7), and G701 (L9) are involved in the mechanism of Pvd-Fe uptake.
Nader
Mirella
M
Métaux et Microorganismes: Chimie, Biologie et Applications, UMR 7175-LC1 Institut Gilbert-Laustriat, CNRS and University Louis Pasteur, ESBS, F-67413 Illkirch, Strasbourg, France.
Dobbelaere
Wim
W
Vincent
Michel
M
Journet
Laure
L
Adams
Hendrik
H
Cobessi
David
D
Gallay
Jacques
J
Schalk
Isabelle J
IJ
D019943:Amino Acid Substitution; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D031541:Fluorescence Resonance Energy Transfer; D007501:Iron / Q000378:metabolism*; D007504:Iron Radioisotopes / Q000378:metabolism; D008958:Models, Molecular; D015394:Molecular Structure; D009842:Oligopeptides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D010957:Plasmids; D011489:Protein Denaturation / Q000187:drug effects; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D017262:Siderophores / Q000737:chemistry / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014508:Urea / Q000494:pharmacology
D001425:Bacterial Outer Membrane Proteins; C082326:FpvA protein, Pseudomonas aeruginosa; D007504:Iron Radioisotopes; D009842:Oligopeptides; D017262:Siderophores; C042453:pyoverdin; D014508:Urea; D007501:Iron; D003545:Cysteine