Function and coordination of the periplasmic pyoverdine maturation proteins

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Pyoverdines are fluorescent siderophores with fundamental relevance for the virulence of pathogenic pseudomonads, such as P. aeruginosa or P. syringae. Also, beneficial host interactions, such as the plant growth promotion by P. fluorescens, are supported by pyoverdines, which contribute to iron supply in iron-limited host environments. Pyoverdines are generated inside the periplasm from ferribactins, which are cytoplasmically non-ribosomally synthesized peptides. All ferribactins initially possess an N-acylated L-glutamate at their N-terminus, followed by a D-tyrosine, L-2,4-diaminobutyrate, and further residues of a variable, strain-specific sequence. Inside the periplasm, the N-terminal glutamate is deacylated and chemically further modified, and the dihydroxyquinoline fluorophore is generated from D-tyrosine and L-2,4-diaminobutyrate, which ultimately results in mature pyoverdine with its high affinity for Fe3+. Secreted pyoverdine chelates Fe3+ with the two oxygen ligands of the fluorophore and functions of the peptide side chains. The iron-complex is taken up into the periplasm, the iron is reductively released and transported into the cytoplasm, and the free pyoverdine is recycled. Six proteins are involved in the periplasmic maturation process: PvdQ (deacylation), PvdP (fluorophore-formation), PvdO (fluorophore-formation), PvdN (decarboxylation and oxygenation of the glutamate), PtaA (transamination of the glutamate) und PvdM (Ferribactin supply for PvdP). Before this project started, only the functions of PvdP and PvdQ were known. In the course of this project, we could reveal the functions of PtaA, PvdO, and PvdM, with PtaA being newly discovered by us. We could also more precisely define the function of PvdP, examine pvdQ and pvdM mutants, and we carried out first biochemical analyses of PvdM. We chose the genome sequenced, genetically and preparatively good amenable S1 organism P. fluorescens A506 as model system, knowing that other fluorescent pseudomonads in principle use the same pyoverdine maturation pathway. In the last years, we have generated in frame deletion mutants for all genes encoding the pyoverdine maturation proteins, and we have generated functional complementation systems, developed expression systems and established the basic methods for the purification and identification of pyoverdines and biogenesis intermediates. With this project, we will now answer more key questions of the periplasmic pyoverdine maturation: We will elucidate the function of PvdQ in more detail, clarify the copper assembly process for PvdP, analyze the redox activity of PvdO, characterize PtaA, and clarify the mechanism of PvdM function. Interaction studies will show, whether substrate handover or channeling mechanisms can prevent the premature loss of intermediates. We are thus very confident to clarify with this project the fascinating and over decades enigmatic process of periplasmic pyoverdine maturation.