Ferric-pyoverdine recognition by Fpv outer membrane proteins of Pseudomonas protegens Pf-5

Abstract

The soil bacterium Pseudomonas protegens Pf-5 (previously called P. fluorescens Pf-5) produces two siderophores, enantio-pyochelin and a compound in the large and diverse pyoverdine family. Using high-resolution mass spectroscopy, we determined the structure of the pyoverdine produced by Pf-5. In addition to producing its own siderophores, Pf-5 also utilizes ferric complexes of some pyoverdines produced by other strains of Pseudomonas spp. as sources of iron. Previously, phylogenetic analysis of the 45 TonB-dependent outer membrane proteins in Pf-5 indicated that six are in a well-supported clade with ferric-pyoverdine receptors (Fpvs) from other Pseudomonas spp. We used a combination of phylogenetics, bioinformatics, mutagenesis, pyoverdine structural determinations, and cross-feeding bioassays to assign specific ferric-pyoverdine substrates to each of the six Fpvs of Pf-5. We identified at least one ferric-pyoverdine that was taken up by each of the six Fpvs of Pf-5. Functional redundancy of the Pf-5 Fpvs was also apparent, with some ferric-pyoverdines taken up by all mutants with a single Fpv deletion but not by a mutant having deletions in two of the Fpv-encoding genes. Finally, we demonstrated that phylogenetically related Fpvs take up ferric complexes of structurally related pyoverdines, thereby establishing structure-function relationships that can be employed in the future to predict the pyoverdine substrates of Fpvs in other Pseudomonas spp.

Fig 1

(A) Homology model of FpvU from P. protegens Pf-5 compared to FpvAI from P. aeruginosa PAO1 showing the structural components, with the β barrel in green, plug in red, N-terminal signaling domain in blue, connecting loop in purple, and TonB box in brown. (B) Positions of amino acid residues in the plug and β-barrel domains of FpvU corresponding to the residues of FpvAI that are involved in pyoverdine binding. Amino acid side chain structures are shown in black. (C) Alignment of FpvAI and FpvU. Amino acid residues of FpvAI that interact with the PAO1 pyoverdine (8, 13) are numbered and shown in red font. Identical residues in FpvU are in red, conservative substitutions are in blue, semiconservative substitutions are in purple, and nonconservative substitutions are in green.

Fig 2

Bioinformatic and structural analysis of the pyoverdine produced by P. protegens Pf-5. (A) Pyoverdine biosynthesis gene clusters of Pf-5 (80). (B) NRPSs in the pyoverdine gene cluster. For each module (M) in the NRPSs, domains (C, condensation; A, adenylation; T, thiolation; E, epimerization; Te, thioesterase) and the specific amino acid predicted from the sequence of each adenylation domain are shown. The NRPS encoded by pvdI, pvdJ, and pvdD synthesizes the peptide chain, and the NRPS encoded by pvdL synthesizes the chromophore. (C) Scheme for the pyoverdine chromophore biosynthesis. The linear tripeptide-intermediate l-Glu–d-Tyr–l-Dab undergoes hydroxylation, dehydration, and cyclization reactions to give the completed quinoline chromophore. The red dot indicates that the absolute configuration at C-1 of the chromophore results from the absolute configuration of the integrated amino acid Dab, which is in turn determined by PvdL. (D) Structure of the Pf-5 pyoverdine determined from high-resolution MS analysis. Suc, succinic acid.

Fig 3

Utilization of a ferric-pyoverdine complex by a siderophore-deficient mutant of Pf-5. Five microliters of an 8 μM solution of pyoverdine B10-2 (from Pseudomonas sp. strain B10) was spotted in the center of each plate containing an iron-limited medium (KMB amended with 600 mM 2,2′-dipyridyl). Ten microliters of a cell suspension (∼10⁶ CFU/ml) of a pvdI-pchA mutant of Pf-5 (Fpv⁺) or the pvdI-pchA mutant with a deletion in one of six fpv genes (fpvU, fpvV, fpvX, fpvY, or fpvZ) was spotted 1 cm from the center of the plate. The plate was incubated at 27°C for 2 days. Growth of all strains with the exception of the pvdI-pchA-fpvW mutant was observed, indicating that FpvW is required for uptake of pyoverdine B10-2.

Fig 4

(A) Neighbor-joining analysis of Fpv proteins. Pf-5 Fpvs are shown in bold boxed font, and Fpvs having known substrates are shown in bold font; TBDPs that take up ferric citrate (FecA) are included as an outgroup. Abbreviations for species represented in the tree are as follows: PFL_, P. protegens Pf-5; Ap ATCC_43553, Achromobacter piechaudii ATCC 43553; Dsp, Delftia sp. strain Cs1-4; Pa PAO1, Pa ATCC 27853, Pa LESB58, Pa UCBPP-PA14, and Pa 7NSK2, P. aeruginosa PAO1, ATCC 27853, LESB58, UCBPP-PA14, and 7NSK2, respectively; Pb NFM421, P. brassicacearum subsp. brassicacearum NFM421; Pc O6 and Pc 30-84, Pseudomonas chlororaphis O6 and 30-84, respectively; Pe 14-3, Pseudomonas extremaustralis 14-3; Pf SBW25, P. fluorescens SBW25; Pp GB-1, Pp KT2440, and Pp WCS358, P. putida GB-1, KT2440, and WCS358, respectively; Psp Ag1, Psp BG33R, Psp GM17, Psp GM24, Psp GM50, Psp GM79, Psp GM102, Psp GM103, and Psp PAMC 25886, Pseudomonas sp. strains Ag1, BG33R, GM17, GM24, GM50, GM79, GM102, GM103, and PAMC 25886, respectively. The consensus tree was inferred from 1,000 replicates, and branches corresponding to partitions reproduced in less than 30% bootstrap replicates were collapsed. The percentage of replicate trees in which the proteins clustered together in the bootstrap test is shown next to each branch. (B) Pyoverdine peptide chain sequences recognized by the adjacent Fpvs. See Table 2 for an explanation of abbreviations and formatting. Pyoverdines associated with Fpvs of Pf-5 in this study (Table 2) are in bold font. Boxes surround structures that are recognized by more than one Fpv in the corresponding clade shown in panel A. The following Fpvs were associated previously with specific ferric-pyoverdine complexes: PupB (40), PupA (74), FpvA (15), FpvB (78), PbuA (73), and FpvAII (47).

Fig 5

Bioinformatic analysis of the NRPSs for the biosynthesis of the pyoverdine peptide chain of P. fluorescens strains SBW25 and A506. For each module (M) in the NRPSs, domains (C, condensation; A, adenylation; T, thiolation; E, epimerization; Te, thioesterase) and the specific amino acid predicted from the sequence of each adenylation domain are shown. The predicted amino acid sequence and composition of the two peptide chains are identical, with the exception of the d versus l configuration of the ornithine residue of module 6 (shown in red font). PvdA and PvdF, which are encoded from the pyoverdine gene clusters of SBW25 (31) and A506 (49), confer the ornithine hydroxylase and ornithine transformylase activities needed to transform ornithine to δN-formyl-δN-hydroxy-ornithine residues.