Amino Acids

, Volume 38, Issue 5, pp 1447–1459 | Cite as

Synthesis of the siderophore pyoverdine in Pseudomonas aeruginosa involves a periplasmic maturation

  • Emilie Yeterian
  • Lois W. Martin
  • Laurent Guillon
  • Laure Journet
  • Iain L. Lamont
  • Isabelle J. Schalk
Original Article


Pyoverdines, the main siderophores produced by fluorescent Pseudomonads, comprise a fluorescent dihydroxyquinoline chromophore attached to a strain-specific peptide. These molecules are thought to be synthesized as non-fluorescent precursor peptides that are then modified to give functional pyoverdines. Using the fluorescent properties of PVDI, the pyoverdine produced by Pseudomonas aeruginosa PAO1, we were able to show that PVDI was not present in the cytoplasm of the bacteria, but large amounts of a fluorescent PVDI precursor PVDIp were stored in the periplasm. Like PVDI, PVDIp is able to transport iron into P. aeruginosa cells. Mutation of genes encoding the periplasmic PvdN, PvdO and PvdP proteins prevented accumulation of PVDIp in the periplasm and secretion of PVDI into the growth medium, indicating that these three enzymes are involved in PVDI synthesis. Mutation of the gene encoding PvdQ resulted in the presence of fluorescent PVDI precursor in the periplasm and secretion of a functional fluorescent siderophore that had different isoelectric properties to PVDI, suggesting a role for PvdQ in the periplasmic maturation of PVDI. Mutation of the gene encoding the export ABC transporter PvdE prevented PVDI production and accumulation of PVDIp in the periplasm. These data are consistent with a model in which a PVDI precursor peptide is synthesized in the cytoplasm and exported to the periplasm by PvdE where siderophore maturation, including formation of the chromophore moiety, occurs in a process involving the PvdN, PvdO, PvdP and PvdQ proteins.


Siderophore biosynthesis Pyoverdine Iron uptake Fluorescence Fluorescent peptide Pseudomonasaeruginosa 



This work was supported by the Centre National de la Recherche Scientifique (CNRS) and by the association Mucoviscidose ABCF. E. Yeterian had a fellowship from the French Ministère de la Recherche et de la Technologie. The authros are grateful to Keith Poole for providing plasmid pJSS2, to Karla Mettrick for constructing the fpvA and fpvA fpvR mutant strains and to Dr Gaëtan L. A. Mislin for mass spectrum analyses and discussion.


  1. Abdallah MA, Pattus F (2000) Siderophores and iron-transport in microorganisms. J Chin Chem Soc 47:1–20Google Scholar
  2. Ackerley DF, Caradoc-Davies TT, Lamont IL (2003) Substrate specificity of the nonribosomal peptide synthetase PvdD from Pseudomonas aeruginosa. J Bacteriol 185:2848–2855CrossRefPubMedGoogle Scholar
  3. Albrecht-Gary AM, Blanc S, Rochel N, Ocacktan AZ, Abdallah MA (1994) Bacterial iron transport: coordination properties of pyoverdin PaA, a peptidic siderophore of Pseudomonas aeruginosa. Inorg Chem 33:6391–6402CrossRefGoogle Scholar
  4. Baysse C, De Vos D, Naudet Y, Vandermonde A, Ochsner U, Meyer JM, Budzikiewicz H, Schafer M, Fuchs R, Cornelis P (2000) Vanadium interferes with siderophore-mediated iron uptake in Pseudomonas aeruginosa. Microbiology 146(Pt 10):2425–2434PubMedGoogle Scholar
  5. Baysse C, Budzikiewicz H, Uria Fernandez D, Cornelis P (2002) Impaired maturation of the siderophore pyoverdine chromophore in Pseudomonas fluorescens ATCC 17400 deficient for the cytochrome c biogenesis protein CcmC. FEBS Lett 523:23–28CrossRefPubMedGoogle Scholar
  6. Braud A, Hoegy F, Jezequel K, Lebeau T, Schalk IJ (2009) New insights into the metal specificity of the Pseudomonas aeruginosa pyoverdine-iron uptake pathway. Environ Microbiol 11:1079–1091CrossRefPubMedGoogle Scholar
  7. Braun V (2003) Iron uptake by Escherichia coli. Front Biosci 8:1409–1421CrossRefGoogle Scholar
  8. Brillet K, Journet L, Celia H, Paulus L, Stahl A, Pattus F, Cobessi D (2007) A β-strand lock-exchange for signal transduction in TonB-dependent transducers on the basis of a common structural motif. Structure 15:1383–1391CrossRefPubMedGoogle Scholar
  9. Budzikiewicz H (2004) Siderophores of the Pseudomonadaceae sensu stricto (fluorescent and non-fluorescent Pseudomonas spp.). Fortschr Chem Org Naturst 87:81–237PubMedGoogle Scholar
  10. Chuanchuen R, Narasaki CT, Schweizer HP (2002) Benchtop and microcentrifuge preparation of Pseudomonas aeruginosa competent cells. Biotechniques 33(760):762–763Google Scholar
  11. Clarke-Pearson MF, Brady SF (2008) Paerucumarin, a new metabolite produced by the pvc gene cluster from Pseudomonas aeruginosa. J Bacteriol 190:6927–6930CrossRefPubMedGoogle Scholar
  12. Clément E, Mesini PJ, Pattus F, Abdallah MA, Schalk IJ (2004) The binding mechanism of pyoverdin with the outer membrane receptor FpvA in Pseudomonas aeruginosa is dependent on its iron-loaded status. Biochemistry 43:7954–7965CrossRefPubMedGoogle Scholar
  13. Cobessi D, Célia H, Folschweiller N, Schalk IJ, Abdallah MA, Pattus F (2005) The crystal structure of the pyoverdin outer membrane receptor FpvA from Pseudomonas aeruginosa at 3.6 Å resolution. J Mol Biol 34:121–134CrossRefGoogle Scholar
  14. Cornelis P, Matthijs S (2002) Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines. Environ Microbiol 4:787–798CrossRefPubMedGoogle Scholar
  15. Demange P, Wendenbaum S, Linget C, Mertz C, Cung MT, Dell A, Abdallah MA (1990) Bacterial siderophores: structure and NMR assignment of pyoverdins PaA, siderophores of Pseudomonas aeruginosa ATCC 15692. Biol Metals 3:155–170CrossRefGoogle Scholar
  16. Dorrestein P, Begley TP (2005) Oxidative cascades: a facile biosynthetic strategy for the assembly of complex molecules. Bioorg Chem 33:136–148CrossRefPubMedGoogle Scholar
  17. Dorrestein PC, Poole K, Begley TP (2003) Formation of the chromophore of the pyoverdine siderophores by an oxidative cascade. Org Lett 5:2215–2217CrossRefPubMedGoogle Scholar
  18. Folschweiller N, Gallay J, Vincent M, Abdallah MA, Pattus F, Schalk IJ (2002) The interaction between pyoverdin and its outer membrane receptor in Pseudomonas aeruginosa leads to different conformers: a time-resolved fluorescence study. Biochemistry 41:14591–14601CrossRefPubMedGoogle Scholar
  19. Fuchs R, Schafer M, Geoffroy V, Meyer JM (2001) Siderotyping—a powerful tool for the characterization of pyoverdines. Curr Top Med Chem 1:31–57CrossRefPubMedGoogle Scholar
  20. Ge L, Seah SY (2006) Heterologous expression, purification, and characterization of an l-ornithine N(5)-hydroxylase involved in pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa. J Bacteriol 188:7205–7210CrossRefPubMedGoogle Scholar
  21. Greenwald J, Hoegy F, Nader M, Journet L, Mislin GLA, Graumann PL, Schalk IJ (2007) Real-time FRET visualization of ferric-pyoverdine uptake in Pseudomonas aeruginosa: a role for ferrous iron. J Biol Chem 282:2987–2995CrossRefPubMedGoogle Scholar
  22. Greenwald J, Nader M, Celia H, Gruffaz C, Geoffroy V, Meyer JM, Schalk IJ, Pattus F (2009) FpvA bound to non-cognate pyoverdines: molecular basis of siderophore recognition by an iron transporter. Mol Microbiol 72:1246–1259CrossRefPubMedGoogle Scholar
  23. Hoang TT, Karkhoff-Schweizer RR, Kutchma AJ, Schweizer HP (1998) A broad-host-range Flp-FRT recombination system for site-specific excision of chromosomally-located DNA sequences: application for isolation of unmarked Pseudomonas aeruginosa mutants. Gene 212:77–86CrossRefPubMedGoogle Scholar
  24. Huang JJ, Petersen A, Whiteley M, Leadbetter JR (2006) Identification of QuiP, the product of gene PA1032, as the second acyl-homoserine lactone acylase of Pseudomonas aeruginosa PAO1. Appl Environ Microbiol 72:1190–1197CrossRefPubMedGoogle Scholar
  25. Imperi F, Putignani L, Tiburzi F, Ambrosi C, Cipollone R, Ascenzi P, Visca P (2008) Membrane-association determinants of the omega-amino acid monooxygenase PvdA, a pyoverdine biosynthetic enzyme from Pseudomonas aeruginosa. Microbiology 154:2804–2813CrossRefPubMedGoogle Scholar
  26. Koebnik R (2005) TonB-dependent trans-envelope signalling: the exception or the rule? Trends Microbiol 13:343–347CrossRefPubMedGoogle Scholar
  27. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM 2nd, Peterson KM (1995) Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-resistance cassettes. Gene 166:175–176CrossRefPubMedGoogle Scholar
  28. Lamont IL, Martin LW (2003) Identification and characterization of novel pyoverdine synthesis genes in Pseudomonas aeruginosa. Microbiology 149:833–842CrossRefPubMedGoogle Scholar
  29. Lamont IL, Beare PA, Ochsner U, Vasil AI, Vasil ML (2002) Siderophore-mediated signaling regulates virulence factor production in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 99:7072–7077CrossRefPubMedGoogle Scholar
  30. Lamont IL, Martin LW, Sims T, Scott A, Wallace M (2006) Characterization of a gene encoding an acetylase required for pyoverdine synthesis in Pseudomonas aeruginosa. J Bacteriol 188:3149–3152CrossRefPubMedGoogle Scholar
  31. Lehoux DE, Sanschagrin F, Levesque RC (2000) Genomics of the 35-kb pvd locus and analysis of novel pvdIJK genes implicated in pyoverdine biosynthesis in Pseudomonas aeruginosa. FEMS Microbiol Lett 190:141–146CrossRefPubMedGoogle Scholar
  32. Lewenza S, Gardy JL, Brinkman FS, Hancock RE (2005) Genome-wide identification of Pseudomonas aeruginosa exported proteins using a consensus computational strategy combined with a laboratory-based PhoA fusion screen. Genome Res 15:321–329CrossRefPubMedGoogle Scholar
  33. Mascarenhas J, Soppa J, Strunnikov AV, Graumann PL (2002) Cell cycle-dependent localization of two novel prokaryotic chromosome segregation and condensation proteins in Bacillus subtilis that interact with SMC protein. EMBO J 21:3108–3118CrossRefPubMedGoogle Scholar
  34. McMorran BJ, Merriman ME, Rombel IT, Lamont IL (1996) Characterisation of the pvdE gene which is required for pyoverdine synthesis in Pseudomonas aeruginosa. Gene 176:55–59CrossRefPubMedGoogle Scholar
  35. McMorran BJ, Kumara HM, Sullivan K, Lamont IL (2001) Involvement of a transformylase enzyme in siderophore synthesis in Pseudomonas aeruginosa. Microbiology 147:1517–1524PubMedGoogle Scholar
  36. Merriman TR, Merriman ME, Lamont IL (1995) Nucleotide sequence of pvdD, a pyoverdine biosynthetic gene from Pseudomonas aeruginosa: PvdD has similarity to peptide synthetases. J Bacteriol 177:252–258PubMedGoogle Scholar
  37. Meyer JM, Stintzi A, De Vos D, Cornelis P, Tappe R, Taraz K, Budzikiewicz H (1997) Use of siderophores to type pseudomonads: the three Pseudomonas aeruginosa pyoverdine systems. Microbiology 143(Pt 1):35–43CrossRefPubMedGoogle Scholar
  38. Meyer JM, Stintzi A, Poole K (1999) The ferripyoverdine receptor FpvA of Pseudomonas aeruginosa PAO1 recognizes the ferripyoverdines of P. aeruginosa PAO1 and P. fluorescens ATCC 13525. FEMS Microbiol Lett 170:145–150CrossRefPubMedGoogle Scholar
  39. Meyer JM, Gruffaz C, Raharinosy V, Bezverbnaya I, Schafer M, Budzikiewicz H (2008) Siderotyping of fluorescent Pseudomonas: molecular mass determination by mass spectrometry as a powerful pyoverdine siderotyping method. Biometals 21:259–271CrossRefPubMedGoogle Scholar
  40. Mossialos D, Ochsner U, Baysse C, Chablain P, Pirnay JP, Koedam N, Budzikiewicz H, Fernandez DU, Schafer M, Ravel J, Cornelis P (2002) Identification of new, conserved, non-ribosomal peptide synthetases from fluorescent pseudomonads involved in the biosynthesis of the siderophore pyoverdine. Mol Microbiol 45:1673–1685CrossRefPubMedGoogle Scholar
  41. Nader M, Dobbelaere W, Vincent M, Journet L, Adams H, Cobessi D, Gallay J, Schalk IJ (2007) Identification of residues of FpvA involved in the different steps of Pvd-Fe uptake in Pseudomonas aeruginosa. Biochemistry 46:11707–11717CrossRefPubMedGoogle Scholar
  42. Ochsner U, Snyder A, Vasil AI, Vasil ML (2002a) Effects of the twin-arginine translocase on secretion of virulence factors, stress response, and pathogenesis. Proc Natl Acad Sci USA 99:8312–8317CrossRefPubMedGoogle Scholar
  43. Ochsner UA, Wilderman PJ, Vasil AI, Vasil ML (2002b) GeneChip expression analysis of the iron starvation response in Pseudomonas aeruginosa: identification of novel pyoverdine biosynthesis genes. Mol Microbiol 45:1277–1287CrossRefPubMedGoogle Scholar
  44. Page WJ, Kwon E, Cornish AS, Tindale AE (2003) The csbX gene of Azotobacter vinelandii encodes an MFS efflux pump required for catecholate siderophore export. FEMS Microbiol Lett 228:211–216CrossRefPubMedGoogle Scholar
  45. Poole K, McKay GA (2003) Iron acquisition and its control in Pseudomonas aeruginosa: many roads lead to Rome. Front Biosci 8:d661–d686CrossRefPubMedGoogle Scholar
  46. Poole K, Heinrichs DE, Neshat S (1993a) Cloning and sequence analysis of an EnvCD homologue in Pseudomonas aeruginosa: regulation by iron and possible involvement in the secretion of the siderophore pyoverdine. Mol Microbiol 10:529–544CrossRefPubMedGoogle Scholar
  47. Poole K, Neshat S, Krebes K, Heinrichs DE (1993b) Cloning and nucleotide sequence analysis of the ferripyoverdine receptor gene fpvA of Pseudomonas aeruginosa. J Bacteriol 175:4597–4604PubMedGoogle Scholar
  48. Postle K, Kadner RJ (2003) Touch and go: tying TonB to transport. Mol Microbiol 49:869–882CrossRefPubMedGoogle Scholar
  49. Ravel J, Cornelis P (2003) Genomics of pyoverdine-mediated iron uptake in pseudomonads. Trends Microbiol 11:195–200PubMedGoogle Scholar
  50. Rombel IT, McMorran BJ, Lamont IL (1995) Identification of a DNA sequence motif required for expression of iron-regulated genes in pseudomonads. Mol Gen Genet 246:519–528CrossRefPubMedGoogle Scholar
  51. Royle PL, Matsumoto H, Holloway BW (1981) Genetic circularity of the Pseudomonas aeruginosa PAO chromosome. J Bacteriol 145:145–155PubMedGoogle Scholar
  52. Sambrook J, Russel DW (2001) Molecular cloning: a laboratory manual. Cold Spring Harbor, NYGoogle Scholar
  53. Saurin W, Hofnung M, Dassa E (1999) Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters. J Mol Evol 48:22–41CrossRefPubMedGoogle Scholar
  54. Schalk IJ (2008) Metal trafficking via siderophores in Gram-negative bacteria: specificities and characteristics of the pyoverdine pathway. J Inorg Biochem 102:1159–1169CrossRefPubMedGoogle Scholar
  55. Schalk IJ, Kyslik P, Prome D, van Dorsselaer A, Poole K, Abdallah MA, Pattus F (1999) Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron transport. Biochemistry 38:9357–9365CrossRefPubMedGoogle Scholar
  56. Schalk IJ, Hennard C, Dugave C, Poole K, Abdallah MA, Pattus F (2001) Iron-free pyoverdin binds to its outer membrane receptor FpvA in Pseudomonas aeruginosa: a new mechanism for membrane iron transport. Mol Microbiol 39:351–360CrossRefPubMedGoogle Scholar
  57. Schalk IJ, Abdallah MA, Pattus F (2002) Recycling of pyoverdin on the FpvA receptor after ferric pyoverdin uptake and dissociation in Pseudomonas aeruginosa. Biochemistry 41:1663–1671CrossRefPubMedGoogle Scholar
  58. Shen J, Meldrum A, Poole K (2002) FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa. J Bacteriol 184:3268–3275CrossRefPubMedGoogle Scholar
  59. Shirley M, Lamont IL (2009) Role of TonB1 in pyoverdine-mediated signaling in Pseudomonas aeruginosa. J Bacteriol 191:5634–5640CrossRefPubMedGoogle Scholar
  60. Simon R, O’Connell M, Labes M, Puhler A (1986) Plasmid vectors for the genetic analysis and manipulation of rhizobia and other gram-negative bacteria. Methods Enzymol 118:640–659CrossRefPubMedGoogle Scholar
  61. Takase H, Nitanai H, Hoshino K, Otani T (2000) Impact of siderophore production on Pseudomonas aeruginosa infections in immunosuppressed mice. Infect Immun 68:1834–1839CrossRefPubMedGoogle Scholar
  62. Vandenende CS, Vlasschaert M, Seah SY (2004) Functional characterization of an aminotransferase required for pyoverdine siderophore biosynthesis in Pseudomonas aeruginosa PAO1. J Bacteriol 186:5596–5602CrossRefPubMedGoogle Scholar
  63. Visca P (2004) Iron regulation and siderophore signalling in virulence by Pseudomonas aeruginosa. In: Juan-Luis R (ed) Pseudomonas, vol 2. Kluwer/Plenum Publishers, New York, pp 69–123Google Scholar
  64. Visca P, Ciervo A, Orsi N (1994) Cloning and nucleotide sequence of the pvdA gene encoding the pyoverdin biosynthetic enzyme l-ornithine N5-oxygenase in Pseudomonas aeruginosa. J Bacteriol 176:1128–1140PubMedGoogle Scholar
  65. Visca P, Leoni L, Wilson MJ, Lamont IL (2002) Iron transport and regulation, cell signalling and genomics: lessons from Escherichia coli and Pseudomonas. Mol Microbiol 45:1177–1190CrossRefPubMedGoogle Scholar
  66. Visca P, Imperi F, Lamont IL (2007) Pyoverdine siderophores: from biogenesis to biosignificance. Trends Microbiol 15:22–30CrossRefPubMedGoogle Scholar
  67. Voulhoux R, Filloux A, Schalk IJ (2006) Role of the TAT System in the pyoverdine-mediated iron acquisition in Pseudomonas aeruginosa. J Bacteriol 188:3317–3323CrossRefPubMedGoogle Scholar
  68. West SE, Schweizer HP, Dall C, Sample AK, Runyen-Janecky LJ (1994) Construction of improved Escherichia–Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81–86CrossRefPubMedGoogle Scholar
  69. Wiener MC (2005) TonB-dependent outer membrane transport: going for Baroque? Curr Opin Struct Biol 15:394–400CrossRefPubMedGoogle Scholar
  70. Yoder MF, Kisaalita WS (2006) Fluorescence of pyoverdin in response to iron and other common well water metals. J Environ Sci Health A Tox Hazard Subst Environ Eng 41:369–380PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Emilie Yeterian
    • 1
  • Lois W. Martin
    • 2
  • Laurent Guillon
    • 1
  • Laure Journet
    • 1
  • Iain L. Lamont
    • 2
  • Isabelle J. Schalk
    • 1
  1. 1.Métaux et Microorganismes, Chimie, Biologie et ApplicationsFRE 3211, CNRS-Université de StrasbourgStrasbourgFrance
  2. 2.Department of BiochemistryUniversity of OtagoDunedinNew Zealand

Personalised recommendations