Mutational Analysis of a Bifunctional Ferrisiderophore Receptor and Signal-Transducing Protein from Pseudomonas aeruginosa

Growth of bacteria.Bacteria were grown at 37°C using L broth and L agar (Gibco BRL) for E. coli and King's B broth and agar (23) for the growth of P. aeruginosa, unless otherwise stated. Antibiotics were added as required at the following concentrations for work with E. coli, with the concentrations used for P. aeruginosa shown in parentheses: ampicillin, 50 μg/ml; chloramphenicol, 30 μg/ml (200 μg/ml); gentamicin, 4 μg/ml (20 μg/ml); and kanamycin, 50 μg/ml.

Linker insertion mutagenesis of fpvA.The fpvA gene from P. aeruginosa PAO1 was amplified from pRV2 (37) by PCR using Expand DNA polymerase (Roche Molecular Biochemicals) with primers fpvA1 (5′-GAGCTCGAAGAGCAATCACCCAT-3′) and fpvA2 (5′-AAGCTTGGCGTTCTTTTTCGCA-3′) that contain synthetic SacI and HindIII restriction sites (shown in bold). The PCR product was treated with SacI and HindIII restriction enzymes and cloned into plasmid pUCP22 (48) using standard methods (42). In the resulting plasmid (pUCP22::fpvA) the fpvA gene is expressed from the E. coli lac promoter.

The cloned DNA was sequenced and the sequence was identical to the wild-type fpvA sequence. pUCP22::fpvA DNA was linearized using restriction enzymes that cut only once in the insert (ClaI, KpnI, and SalI) or by partial digestion with limiting amounts of more frequently cutting enzymes (AluI, EcoRV, Sau3AI, and TacI) in the presence of ethidium bromide (2) and linearized plasmid DNA was purified from agarose gels using the Qiaex II Gel Extraction Kit (Qiagen). The linearized DNA was ligated with kanamycin resistance cassettes that had been purified from agarose gels following treatment of plasmids pUC4KISS (2) and pNRE1 (29) with appropriate restriction enzymes. The ligated DNA was transformed into E. coli MC1061 (9), and bacteria resistant to ampicillin, gentamicin and kanamycin were selected.

The approximate locations of the kanamycin cassette insertions in the resulting plasmids were determined by restriction analysis. The precise locations of insertions in fpvA were determined by sequencing of plasmid DNA using primers Km1 (AGATTTTGAGACACAACG) and Km2 (TTACGCTGACTTGACGGG) that correspond to outward-facing kanamycin cassette sequences in conjunction with an Applied Biosystems Automated sequencer (Centre for Gene Research, University of Otago). The kanamycin cassettes were then excised from the plasmids using a suitable flanking restriction enzyme and the plasmids treated with DNA ligase and transformed into E. coli MC1061 with selection for Ap^r Gm^r Km^s bacteria. This procedure resulted in the insertion of short DNA segments (12 or 24 oligonucleotides) at 36 different sites in fpvA (Table 1). The DNA spanning each insertion site was sequenced to confirm that the expected mutational events had occurred. Plasmids containing wild-type and mutant DNA, as well as a vector-only control, were transformed into the P. aeruginosa fpvA mutant strain K690 fpvA::Tc (32) which has a Tet^r interposon inserted at an internal ScaI site (K. Poole, personal. communication).

Western blotting.Outer membranes were prepared from cells of P. aeruginosa and FpvA was analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting using a polyclonal anti-FpvA serum as described previously (4). Antibodies against the C terminus of FpvA were obtained by conjugating a peptide (GDPRNLMFSTRWDF, corresponding to residues 802 to 815 of the mature protein) with an equal amount (5 mg) of keyhole limpet hemocyanin (Roche) by overnight incubation in the presence of 0.1% glutaraldehyde (19). Following dialysis against phosphate-buffered saline, one tenth of the resulting mixture was mixed with an equal amount of Freund's complete adjuvant and used to immunize a New Zealand White rabbit. Nine booster immunizations were carried out at seven-day intervals after mixing the remainder of the conjugated peptide with an equal volume of Freund's incomplete adjuvant. Three days after the final immunization the animal was bled and serum was recovered from clotted blood. The serum was used in Western blotting in conjunction with goat anti-rabbit immunoglobulin G (whole molecule) horseradish peroxidase conjugate and 3-amino-9-ethylcarbazole (Sigma) as described (19).

To analyze the FpvA protein in whole cells of P. aeruginosa, bacteria were grown in Kings B medium overnight. The optical density at 600 nm (OD₆₀₀) was measured and approximately 1.5 × 10⁹ cells were removed from each culture and collected by centrifugation. The bacterial pellet was resuspended in 10 μl of double-distllled H₂O, 2 μl of 6× SDS-PAGE loading dye was added and the samples were boiled for 5 min. Samples were then electrophoresed on a 10% SDS-PAGE gel for 1 h at 180 V. Protein was transferred to nitrocellulose membrane by Western blotting and FpvA was detected using the polyclonal anti-FpvA serum.

Treatment of FpvA with proteinase K.Overnight cultures (approximately 1.5 × 10⁹ cells) were centrifuged and the pellets were washed in 1 ml of Tris buffer (0.2 M Tris-HCl, 150 mM NaCl, pH 8.0) and then suspended in 1 ml Tris-CaCl₂ buffer (0.2 M Tris-HCl, 150 mM NaCl, 10 mM CaCl₂, pH 8.0). Proteinase K (20 μl; 5 mg ml⁻¹) was added and the samples were incubated for 30 min at 37°C. The reaction was stopped by the addition of 10 μl of 0.2 M phenylmethylsulfonyl fluoride and the cells were spun for 10 min at 13,200 rpm. The cell pellet was then washed with 1 ml Tris buffer supplemented with 10 mM EDTA and 2 mM phenylmethylsulfonyl fluoride.

For digestion of outer membrane preparations, samples were boiled in 0.5 M NaOH and the amount of protein was then assayed (DC protein assay, Bio-Rad). Purified outer membranes containing 50 to 60 μg of protein were suspended in 1 ml of Tris-CaCl₂ buffer, 20 μl of proteinase K was added and the mixture was incubated for 30 min at 37°C. The reaction was stopped by addition of 10 μl of 0.2 M phenylmethylsulfonyl fluoride and the samples were centrifuged for 1 h at 21,000 rpm in a Beckman J2-MC ultracentrifuge. The outer membrane pellet was then washed with 1 ml Tris buffer supplemented with 10 mM EDTA and 2 mM phenylmethylsulfonyl fluoride. The outer membrane and whole-cell samples were resuspended in 30 μl of double-distilled H₂O and placed at −20°C for 1 to 2 h. SDS-PAGE loading dye (5 μl) was added and the samples were boiled for 5 min and SDS-PAGE was carried out.

The sequence of amino acids at the N terminus of a truncated form of FpvA was determined by preparing outer membrane samples, carrying out SDS-PAGE, and transferring protein to a polyvinylidene difluoride membrane (Bio-Rad) with protein being detected by light staining with Ponceau S (41). The position of FpvA was marked, the sample was destained by washing in acetic acid (1%), the band was excised and the N-terminal sequence was determined by the Protein Microchemistry Facility, Department of Biochemistry, University of Otago.

Measurement of ferripyoverdine transport.Pyoverdine was purified from P. aeruginosa strain PAO using the method described (31). Ferripyoverdine uptake assays were performed using a modification of the method described (36). Bacteria were grown in 5 ml of succinate medium (33) to an OD₆₀₀ of 1.0. The cells were collected by centrifugation (7700 g, 4°C, 5 min), washed twice in 5 ml of ice-cold succinate medium and resuspended in the same medium to an OD₆₀₀ of 0.67. Portions of cells (150 μl) were placed in microfuge tubes (1.5 ml) and incubated at 37°C for 15 min. Uptake was initiated by the addition of 55 μl of a solution comprising 50 μl of succinate medium, 4 μl (16 μg) of pyoverdine and 1 μl (24 pmol) of ⁵⁵Fe (Amersham Life Sciences; 1.2 mCi/μmol). Tubes were removed at intervals and the cells collected by centrifugation (30 seconds). The supernatants were immediately aspirated off and the pellets were resuspended in 40 μl of 20% (vol/vol) Triton X-100 and transferred to a scintillation vial. Scintillant (3.75 ml optiPhase Hisafe 2 [Wallac]) was added to the vials and the radioactivity was measured using an LKB Wallac RackBeta liquid scintillation counter; 1 pmol of ⁵⁵Fe gave 2,000 cpm.

Measurement of pyoverdine and FpvA-dependent signaling.Strains of P. aeruginosa were grown until the OD₆₀₀ was between 0.8 and 1.2 and the amount of pyoverdine was measured spectrophotometrically as described previously (30). The amounts of pyoverdine were calculated using the molar extinction coefficient 1.4 × 10⁴ M⁻¹.cm⁻¹ at 405 nm (1) and were normalized for OD₆₀₀. Pyoverdine-mediated signaling was measured using P. aeruginosa K690 fpvA(pMP190::pvdE) (26) that had been transformed with pUCP22-derived plasmids carrying the fpvA derivatives to be tested and that contains the promoter of the pvdE gene upstream of the lacZ reporter gene (40). The amounts of β-galactosidase made by the bacteria were measured as described previously (26) and reflect the ability of the FpvA protein to initiate the signaling process.

Signaling domain of FpvA is likely to lie in the periplasm.FpvA consists of a β-barrel with a plug domain in the outer membrane and an N-terminal signaling domain that is predicted to interact with FpvR in the periplasm (4, 10, 15, 26, 45). To test whether this part of FpvA is located in the periplasm, purified outer membranes and whole cells of P. aeruginosa were treated with proteinase K. FpvA in whole cells was unaffected by proteinase K treatment, whereas treatment of outer membranes resulted in FpvA's being shortened to about 75 kDa (Fig. 1A). Serum raised against a 12-residue peptide corresponding to the C terminus of FpvA also detected the truncated form of FpvA obtained following treatment of outer membranes with proteinase K, indicating that this treatment had removed the N terminus of FpvA (Fig. 1B). These data are consistent with at least part of the 12-kDa N-terminal portion of FpvA being exposed at the periplasmic face of the outer membrane, as expected. Consistent with this result, the N-terminal region of FpvA was degraded in the absence of proteinase inhibitors during the purification of FpvA, suggesting that this part of FpvA is prone to proteolysis (43), most likely because it is not within the outer membrane.

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FIG. 1.

Subcellular localization of the N-terminal region of FpvA. Intact cells and outer membranes of wild-type P. aeruginosa were incubated with (+) or without (−) proteinase K for 1 h. Proteins were then separated by SDS-PAGE and FpvA was detected by Western blotting using a polyclonal anti-FpvA antibody (A) or an antibody specific for the C terminus of FpvA (B). The positions of molecular size markers are indicated. OM, outer membranes; W/C, whole cells.

Mutagenesis of fpvA and incorporation of mutant proteins into the outer membrane.Linker insertion mutagenesis was performed on pUCP22::fpvA as described in Materials and Methods. This method allowed the incorporation of 12- or 24-bp DNA segments into fpvA, resulting in four- or eight-amino-acid insertions at 36 different sites within the FpvA protein. The mutations were scattered throughout the fpvA gene (Fig. 2) and are listed in Table 1.

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FIG. 2.

Locations of mutations in FpvA. The predicted boundaries of domains of FpvA were identified by aligning the FpvA sequence with those of FepA, FhuA, and FecA. The positions of mutations obtained in this study that allowed detection of FpvA by Coomassie staining (▾), reduced amounts of FpvA so that it could only be detected by Western blotting (dotted triangle) or prevented incorporation (▿) (Fig. 3 and Table 1) are shown. Mutations identified previously (21) as permitting (•) or preventing (○) incorporation of FpvA into the outer membrane are also shown. Numbering is relative to the first amino acid residue of mature FpvA (37).

Plasmids carrying the mutated genes were transformed into strain K690, an fpvA mutant of P. aeruginosa, and the presence of FpvA in the outer membranes of the bacteria was assessed following purification of outer membranes and SDS-PAGE gel electrophoresis. Mutant proteins that were not clearly detected by Coomassie staining were also analyzed by Western blotting. Twenty-seven of the mutant proteins were detected by Coomassie staining (Fig. 3A and B; Table 2), indicating that the mutations did not prevent incorporation of the protein into the outer membrane. Four mutations (L55, N224, V401, and G576) resulted in proteins that could only be detected by Western blotting (Fig. 3C), with the L55 protein being smaller than the wild-type protein. The remaining five mutations (A-12, P257, E294, L564, and A724) resulted in no FpvA protein being detectable in the outer membrane fraction. These strains were examined for the presence of the mutant protein in the cells by Western blotting (Fig. 3D). The five mutant proteins that could not be detected in outer membranes also could not be detected in cells, indicating that they were not synthesized or were rapidly degraded.

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FIG. 3.

Production and membrane incorporation of mutant FpvA proteins. A and B. Detection of FpvA by Coomassie staining. Outer membranes were prepared from P. aeruginosa containing wild-type fpvA or the mutant alleles shown and analyzed by SDS-PAGE followed by Coomassie staining. The position of FpvA is indicated. C. Detection of FpvA in outer membranes by Western blotting. Outer membrane samples were analyzed by Western blotting using a polyclonal antibody against FpvA. D. Detection of FpvA in whole cells. Protein was prepared from whole cells of P. aeruginosa and analyzed by Western blotting.

Mutations in the signaling domain of FpvA (R21, I25, L46, and L55) resulted in the presence of truncated FpvA as well as full-size FpvA in the outer membrane (Fig. 3C). This suggested that these mutations affected the structure of the signaling domain so that it was more susceptible to proteolysis. The sequence at the N terminus of the truncated FpvA resulting from the insertion at L46 was determined in order to locate the site of the truncation in this mutant. The sequence was found to be EAXDS, where the identity of the third amino acid could not be determined. This sequence corresponds to residues 76 to 80 of mature FpvA and is consistent with the size (78 kDa) of the truncated FpvA arising from the L46 mutation.

Effects of mutations on ferripyoverdine transport. P. aeruginosa K690 containing the mutated fpvA genes was tested for the ability to transport ferripyoverdine using a time course assay (36). The relative amounts of iron acquired by different mutants are shown in Table 2. As expected, mutations that resulted in the absence of detectable FpvA in the outer membrane also reduced uptake of ferripyoverdine to levels that were not significantly different from those of the FpvA⁻ mutant. The residual ferripyoverdine transport observed with these strains may be due to a second ferripyoverdine transporter, FpvB (17).

Fourteen of the remaining 31 mutations resulted in uptake of ferripyoverdine at rates comparable to those of the FpvA mutant and a further nine resulted in levels of uptake above those of the FpvA mutant but below those seen with wild-type FpvA. Only three of the mutations in the plug or barrel regions of FpvA (L297, P475, and L685) did not have a major effect on ferripyoverdine transport. None of the five mutations in the signaling domain (E4 to L55) affected ferripyoverdine transport.

Effects of the mutations on production of pyoverdine and expression of pyoverdine synthesis genes.FpvA is part of a signaling system that controls production of pyoverdine by P. aeruginosa, and consistent with this, deletion of fpvA or its signaling domain results in reduced expression of pyoverdine synthesis genes and reduced production of pyoverdine (26, 45). The amounts of pyoverdine made by P. aeruginosa carrying the mutant fpvA alleles were determined. All five of the mutations in the signaling domain (E4 to L55) resulted in reduced pyoverdine production. Most of the remaining mutations also resulted in reduced pyoverdine synthesis. This included mutations (L297, P475, and L685) that had only a small effect on transport of ferripyoverdine (Table 2). Strikingly, mutations D510, Y511, and E594 that abolished the transport activity of FpvA did not reduce pyoverdine synthesis.

The pyoverdine signaling system involves posttranslational control of the activity of the alternative sigma factor PvdS by the pyoverdine-FpvA-FpvR signaling pathway (26). It was likely that the effects of mutations in fpvA on pyoverdine synthesis were due to changes in the expression of pyoverdine synthesis genes that are controlled by this pathway. To test this, the effects of selected mutations in fpvA on expression of a representative pyoverdine synthesis gene (pvdE) was measured using a pvdE::lacZ fusion. The amount of expression of lacZ from this construct was a measure of the ability of FpvA to initiate the signaling process. As expected, expression of pvdE::lacZ correlated well with the amount of pyoverdine that was made by P. aeruginosa containing different alleles of fpvA, with the T90 mutation being the only clear exception (Table 2).