FpvA receptor involvement in pyoverdine biosynthesis in Pseudomonas aeruginosa

Abstract

Alignment of the Pseudomonas aeruginosa ferric pyoverdine receptor, FpvA, with similar ferric-siderophore receptors revealed that the mature protein carries an extension of ca. 70 amino acids at its N terminus, an extension shared by the ferric pseudobactin receptors of P. putida. Deletion of fpvA from the chromosome of P. aeruginosa reduced pyoverdine production in this organism, as a result of a decline in expression of genes (e.g., pvdD) associated with the biosynthesis of the pyoverdine peptide moiety. Wild-type fpvA restored pvd expression in the mutant, thereby complementing its pyoverdine deficiency, although a deletion derivative of fpvA encoding a receptor lacking the N terminus of the mature protein did not. The truncated receptor was, however, functional in pyoverdine-mediated iron uptake, as evidenced by its ability to promote pyoverdine-dependent growth in an iron-restricted medium. These data are consistent with the idea that the N-terminal extension plays a role in FpvA-mediated pyoverdine biosynthesis in P. aeruginosa.

FIG. 1.

Multiple-sequence alignment of FpvA and its homologues. Proteins related to FpvA were identified with BLAST (1) (http://www.ncbi.nlm.nih.gov/BLAST/), and the mature sequences (putative signal peptidase cleavage sites were identified by the method of Nielsen et al. [46] [http://www.cbs.dtu.dk/services/SignalP/]) were aligned with CLUSTALW (68) (http://www.ebi.ac.uk/clustalw/). Only the N termini are shown. FptA, P. aeruginosa Fe(III)-pyochelin receptor (accession number P42512); PbuA, P. putida M114 Fe(III)-pseudobactin M114 receptor (accession number Q08017); FhuE, E. coli receptor for Fe(III)-coprogen, -ferrioxamine B, and -rhodotorulic acid (accession number P16869); FauA, Bordetella pertussis Fe(III)-alcaligin receptor (accession number AAD26430); PupA, P. putida WCS358 Fe(III)-pseudobactin 358 receptor (accession number P25184); FpvA, P. aeruginosa Fe(III)-pyoverdine receptor (accession number P48632); PupB, P. putida WCS358 Fe(III)-pseudobactin BN7/BN8 receptor (accession number P38047); PA2466, probable P. aeruginosa TonB-dependent receptor (accession number D83337).

FIG. 2.

Pyoverdine production by P. aeruginosa strains K767 (•), K1660 (▴), K1660(pRK415) (□), K1660(pJSS4) (○), K1660(pJSS5) (▪), K1661 (▾), and K1662 (▵) as a function of growth in iron-deficient succinate minimal medium. Pyoverdine levels were estimated by measuring the A₄₀₅ of the cell-free culture supernatant (A) and hydroxamate nitrogen at A₅₂₀ (B). Values are normalized with respect to culture density, so as to provide a measure of per-cell pyoverdine production. All strains grew equally well in the minimal medium, plateauing at an A₆₀₀ of ca. 1.4 after 7 h of growth. The results are representative of three independent experiments run on different days. Note that data points for K1662 in panel A are masked by data points of other strains in the lower part of the graph.

FIG. 3.

Growth of P. aeruginosa strains K767 (•), K1660 (▴), K1660(pRK415) (□), K1660(pJSS4) (○), K1660(pJSS5) (▪), K1661 (▾), and K1662 (▵) in EDDHA-supplemented (3 mg/ml) succinate minimal medium. The results are representative of three independent experiments run on different days.

FIG. 4.

rpsL (A) and pvdD (B) expression in P. aeruginosa cultured under iron-sufficient (odd-numbered lanes) and iron-deficient (even-numbered lanes) conditions measured by RT-PCR of total RNA (24 ng, top of panel A; 4 ng, bottom of panel A; 4 ng, panel B) isolated from strains K767 (lanes 1 and 2), K1660 (lanes 3 and 4), K1661 (lanes 5 and 6), and K1662 (lanes 7 and 8).

FIG. 5.

Western immunoblot showing FpvA production in P. aeruginosa strains K767 (lane 1), K1660 (lane 2), K1660(pRK415) (lane 3), K1660(pJSS4) (lane 4), K1661 (lane 5), K1660(pJSS5) (lane 6), and K1662 (lane 7) grown in iron-limited succinate minimal medium. Whole-cell extracts were prepared from stationary-phase cells and probed with antibodies to FpvA following electrophoresis and electroblotting as described in Materials and Methods.

FIG. 6.

Influence of exogenously added pyoverdine on growth of P. aeruginosa strains K767 (circles), K1660 (triangles), and K1662 (squares) in EDDHA-supplemented (1.5 mg/ml) succinate minimal medium. Filled symbols, no pyoverdine added; open symbols, 100 μg of pyoverdine added per ml.