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J Bacteriol. Sep 2009; 191(18): 5634–5640.
Published online Jul 10, 2009. doi:  10.1128/JB.00742-09
PMCID: PMC2737950

Role of TonB1 in Pyoverdine-Mediated Signaling in Pseudomonas aeruginosa[down-pointing small open triangle]

Abstract

Pyoverdines are siderophores secreted by Pseudomonas aeruginosa. Uptake of ferripyoverdine in P. aeruginosa PAO1 occurs via the FpvA receptor protein and requires the energy-transducing protein TonB1. Interaction of (ferri)pyoverdine with FpvA activates pyoverdine gene expression in a signaling process involving the cytoplasmic-membrane-spanning anti-sigma factor FpvR and the sigma factor PvdS. Here, we show that mutation of a region of FpvA that interacts with TonB1 (the TonB box) prevents this signaling process, as well as inhibiting bacterial growth in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid). Signaling via wild-type FpvA was also eliminated in strains lacking TonB1 but was unaffected in strains lacking either (or both) of two other TonB proteins in P. aeruginosa, TonB2 and TonB3. An absence of pyoverdine-mediated signaling corresponded with proteolysis of PvdS. These data show that interactions between FpvA and TonB1 are required for (ferri)pyoverdine signal transduction, as well as for ferripyoverdine transport, consistent with a mechanistic link between the signaling and transport functions of FpvA.

Pseudomonas aeruginosa is an opportunistic pathogen that is able to cause severe infections in patients with cystic fibrosis and in immunocompromised individuals, such as burn victims. Under conditions of iron limitation, P. aeruginosa secretes an iron-scavenging compound (siderophore) called pyoverdine. Ferripyoverdine is transported back into the bacteria by an outer membrane (OM) receptor protein, FpvA. The transport of ferripyoverdine via FpvA requires energy provided by a TonB complex (36, 42, 50). TonB is an energy-transducing protein that couples the energy of the cytoplasmic membrane (CM) to a variety of OM receptors required for the import of ferrisiderophores and other molecules. TonB acts in a complex with two CM-associated proteins, ExbB and ExbD, both of which are required for full TonB function (5, 37). The TonB-ExbB-ExbD complex has been identified in many gram-negative bacterial species and is thought to be a conserved mechanism for energy transduction to OM receptor proteins (31). TonB-dependent receptors contain a conserved protein motif known as the TonB box (5). Direct interaction between TonB and the TonB box has been demonstrated for several TonB-dependent receptors (8, 26, 33, 35, 47). Mutations of the TonB box, particularly mutations that are likely to affect the secondary structure, can result in a TonB-uncoupled phenotype characterized by loss of TonB-dependent functions (ferrisiderophore transport) with no loss of TonB-independent functions, such as internalization of bacteriophage (37).

The P. aeruginosa PAO1 genome contains three tonB genes, tonB1 (PA5531) (36), tonB2 (PA0197) (55), and tonB3 (PA0406) (20), encoding proteins of 342, 270, and 319 amino acids (aa), respectively. The TonB1 and TonB2 amino acid sequences display 31% identity over a section of 187 aa, but otherwise, the three PAO1 TonB proteins show similarity (30 to 40% aa identity) to each other only over short (<70-aa) regions. TonB1 is considered to be the primary TonB protein involved in iron transport in P. aeruginosa. tonB1 mutants are impaired for growth in iron-limited medium and are defective for siderophore-mediated iron transport and heme utilization (36, 50, 55). Moreover, direct interaction between TonB1 and the ferripyoverdine receptor FpvA has been demonstrated in vitro (1). The tonB2 gene is not required for growth in iron-limited medium (55). However, tonB1 tonB2 double mutants grow even less well under iron limitation than tonB1 mutants, indicating that TonB2 may be able to partially complement TonB1 in its role in iron acquisition (55). The tonB3 gene is required for twitching motility and assembly of extracellular pili (20), but it is not known whether TonB3 has a role in iron acquisition. Genes encoding ExbB and ExbD proteins are located directly downstream of tonB2 (55) but are not found in association with tonB1 or tonB3.

Besides its role in ferripyoverdine transport, FpvA is part of a signal transduction pathway and thus belongs to a subset of TonB-dependent receptors known as TonB-dependent transducers (reviewed in references 23 and 51). Mutational analysis has shown that the ferripyoverdine transport and signaling roles of FpvA are separate and discrete functions (21, 46). Besides FpvA, the signal transduction pathway involves a CM-spanning anti-sigma factor protein, FpvR, and (ferri)pyoverdine. (It was previously thought that both ferri- and apopyoverdine could bind FpvA (43). However, it was recently reported that only ferripyoverdine is able to form a high-affinity interaction with FpvA (13). The designation (ferri)pyoverdine will be used here to represent the active signaling molecule. FpvA and (ferri)pyoverdine regulate the activity of FpvR, which in turn regulates the activities of two extracytoplasmic function family sigma factors, PvdS and FpvI (3, 25). Upon binding of (ferri)pyoverdine to FpvA, a signal is transmitted to FpvR, resulting in activation of PvdS and FpvI. Activation of PvdS is required for maximal synthesis of pyoverdine itself, as well as two secreted proteins (25). Activation of FpvI leads to increased expression of fpvA (3, 39). In the absence of pyoverdine-mediated signaling, caused by the lack of FpvA or pyoverdine or overexpression of FpvR, suppression of PvdS- and FpvI-dependent gene expression occurs (3, 25), and this is associated with proteolysis of PvdS (49).

Analogous siderophore transport and signaling systems involving an OM TonB-dependent transducer, a CM-bound anti-sigma factor, and an extracytoplasmic function family sigma factor have been described in other bacteria, including the ferric citrate (Fec) system in Escherichia coli and the pseudobactin (Pup) system in Pseudomonas putida (reviewed in reference 6). The TonB protein is required for signaling in both the Fec (14, 33) and Pup (24) systems. Similarly, a TonB system is required for hemophore transport and signaling in Serratia marcescens (4). The aim of this study was to investigate whether TonB was required for pyoverdine-mediated signaling in P. aeruginosa, and if so, to identify which of the three TonB proteins was involved.

MATERIALS AND METHODS

Bacterial strains and growth conditions.

The P. aeruginosa strains used in this study are listed in Table Table1.1. All strains were maintained on Luria Bertani (LB) agar. For tonB mutants, FeCl3 (100 μM) was added to the agar to assist growth. The bacteria were grown at 37°C; broth cultures were aerated by shaking (260 rpm). Antibiotics were included in media as appropriate at the following concentrations: for E. coli, 100 μg/ml ampicillin and 5 μg/ml tetracycline; for P. aeruginosa, 7 μg/ml tetracycline, 50 to 300 μg/ml chloramphenicol, and 25 μg/ml gentamicin. DNA manipulations were performed according to standard methods (41). Pyoverdine, purified from P. aeruginosa PAO1 (29), and desferrioxamine (Sigma-Aldrich) were added to cultures to a final concentration of 60 μM where stated. Pyoverdine-deficient tonB1 (K1040) (36, 55), tonB2 (K1407) (55), and tonB1 tonB2 (K1408) (55) mutants were generously provided by K. Poole (Queen's University, Canada).

TABLE 1.
P. aeruginosa strains used in this study

Construction of ΔfpvA mutant strains.

P. aeruginosa PAO fpvA and PAO pvdF fpvA strains, in which the entire fpvA gene had been deleted, were constructed using pJSS2 as described previously (46); the latter strain also has a mutation in the pvdF gene that is required for pyoverdine synthesis (27). Deletion of fpvA was confirmed using PCR with primer A (5′-CAGGGCAAGCAGGCTTCG-3′) and pvdDfor (5′-TGCTGATGCTCAAGGAGCGG-3′), which flank the deletion.

Disruption of the FpvA TonB box.

The TonB box of FpvA, represented by residues 129 to 135 of the unprocessed FpvA protein (11, 54), was changed from ATMITSN to ATAAAAN using overlap extension PCR (15, 16). PCR was first performed using the primer pairs FpvApromF (5′-AAAGGATCCGGCAAGACCATCATCGTGA-3′) with TonBoxmutR (5′-GGCCGCTGCAGCGGTGGCGCCGAGATCGAC-3′) and TonBoxmutF (5′-GCTGCAGCGGCCAACCAGTTGGGCACCAT-3′) with FpvAmidR (5′-GACGTCCAGTTCGGAGCGGTAA-3′), using P. aeruginosa PAO1 genomic DNA as a template. The resulting PCR products were mixed, and a second PCR was performed using primers FpvApromF and FpvAmidR. The product of this reaction was digested with BamHI, which cuts at an introduced site (underlined) in FpvApromF, and EcoRI, which cuts at a naturally occurring site in fpvA. The resulting product was ligated with the 1.6-kb EcoRI-HindIII fragment of pUCP22::fpvA (21) and cloned into the BamHI-HindIII sites of pUCP22 (52) to give pUCP22::fpvATBM. The wild-type fpvA gene, including the promoter region, was cloned in pUCP22 by ligating the 1.6-kb EcoRI-HindIII fragment of pUCP22::fpvA to a fragment amplified from PAO1 genomic DNA using PCR primers FpvApromF and FpvAmidR and digested with BamHI and EcoRI. This construct was designated pUCP22::fpvAWT. The cloned DNA of pUCP22::fpvAWT and pUCP22::fpvATBM was sequenced to confirm the absence of any other mutations and then subcloned into the integration vector mini-CTX2 (18). Mini-CTX2::fpvAWT and mini-CTX2 fpvATBM were introduced into E. coli S17-1 (48) via transformation (28) and selection on LB agar with tetracycline. The plasmids were then introduced by conjugation (18) into Pseudomonas PAO fpvA and PAO pvdF fpvA. Selection for transconjugants was on LB agar with tetracycline, to select for the presence of mini-CTX plasmids that had integrated at the attP attachment site, and kanamycin (50 μg/ml), to counterselect the S17-1 donor. Vector backbone DNA was removed using the Flp recombinase procedure (17). The resulting strains carried the fpvA gene (either wild type or containing the TonB box mutation) on the chromosome in single copies under the control of the native fpvA promoter. PCR was performed with primers FpvAmidF (5′-GACCGGGTGGAAGTACTCAA-3′) and FpvAmidR2 (5′-CCCGAGATTTCCGCCTCGTA-3′) to demonstrate the presence of the fpvA gene (Fig. (Fig.1B1B).

FIG. 1.
PAO fpvA mutants expressing FpvA with an altered TonB box. (A) Deletion of fpvA. PCR was carried out with primer A and pvdDfor, which flank fpvA. Amplification of a 1.6-kb fragment indicated deletion of a 2.4-kb genomic fragment containing the entire ...

Growth assays.

To assay the growth of strains in the presence of the iron-chelating compound ethylenediamine-di(o-hydroxy-phenylacetic acid) (EDDHA), cultures grown overnight in King's B medium (22) were subcultured into King's B medium containing EDDHA (200 μg/ml) (BDG Synthesis) to an optical density at 600 nm (OD600) of ~0.01. Growth was monitored by measuring the OD600. Growth assays in iron-deficient succinate minimal medium were performed as described by Zhao and Poole (55).

Measurement of pyoverdine production.

Strains were grown in King's B medium to stationary phase (24 h). The absorbances of culture supernatants were measured at OD403, a wavelength at which pyoverdine has a characteristic peak (19).

Promoter activity assays.

Pyoverdine-mediated signaling was assessed by measuring PvdS-dependent gene expression using the reporter plasmid pMP190::PpvdE (40, 53). In this plasmid, expression of a lacZ reporter gene is under the control of the PvdS-dependent pvdE promoter. pMP190::PpvdE was introduced into appropriate strains via transformation (10). For the assays, overnight cultures grown in King's B medium containing chloramphenicol (50 μg/ml) were diluted in King's B medium to an OD600 of ~0.1. Duplicate cultures were grown to an OD600 of ~0.8, and then pyoverdine (60 μM) was added to one of the duplicates. Incubation was continued for a further 2 h, and β-galactosidase assays were performed according to the method of Miller (30).

OM preparations.

Cultures were grown as described for the promoter activity assays with pyoverdine (except chloramphenicol was omitted from the overnight cultures), and OMs were prepared using the sarcosyl solubilization method of Filip et al. (12) as described previously (3). Protein samples (~2 μg) were electrophoresed on a 6% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and submitted to Western analysis with a polyclonal anti-FpvA antibody as described previously (3).

Construction of ΔtonB3 mutant strains.

Mutants containing an in-frame deletion of tonB3 were engineered using the gene replacement vector pEX18Gm (17). A 0.8-kb region of DNA upstream of tonB3 and a 0.9-kb region of DNA downstream of tonB3 were amplified from P. aeruginosa PAO1 genomic DNA using the PCR primer pairs TB3UF (5′-AAAGGATCCGACCAACCGCTGCACGAACT-3′) with TB3UR (5′-AAACTGCAGATGACGCACTCCGCGCCTTG-3′) and TB3DF (5′-CCCCTGCAGCGGCTGTCCAGCAAGTAGCG-3′) with TB3DR (5′-AAAAAGCTTCTTGGGCTGCCGTCCATGTT-3′), and the products were cloned in pUC19 following digestion at introduced restriction sites (underlined). DNA sequencing confirmed that the cloned DNA was free of mutations. The cloned tonB3 flanking regions were ligated together at the common PstI site and cloned into the BamHI-HindIII sites of pEX18Gm to give pEX18Gm::ΔtonB3 containing the tonB3 flanking DNA with a 933-bp internal deletion in tonB3. pEX18Gm::ΔtonB3 was introduced into P. aeruginosa strains PAO6609 (Pvd) (19), K1040, K1407, and K1408 via conjugation (as described above) with selection on LB agar with FeCl3 (100 μM), gentamicin, and kanamycin (50 μg/ml). Secondary recombination causing deletion of the tonB3 gene and removal of vector DNA was selected by sucrose counterselection as described previously (17, 50). Deletion of tonB3 was confirmed using PCR with primers TB3UF and TB3DR (data not shown).

Western analysis of PvdS.

Western analysis was carried out as described previously (49). Briefly, cultures were grown to late exponential phase in King's B medium with or without pyoverdine (60 μM). Cells (1.1 × 109 CFU) were lysed by boiling them (5 min), and samples (~15 μg of protein) were electrophoresed on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels. Western analysis was performed with an anti-PvdS monoclonal antibody (mAbI308).

RESULTS AND DISCUSSION

The TonB box is required for interactions between TonB proteins and TonB-dependent receptors (35, 38, 47), and the position of the TonB box in the structure of FpvA is consistent with this function (7, 11, 44). P. aeruginosa strains were constructed in which fpvA was deleted and replaced with wild-type fpvA or fpvATBM, in which the TonB box of FpvA, represented by residues 129 to 135 of the unprocessed FpvA protein (11, 54), is changed from ATMITSN to ATAAAAN (Fig. 1A and B). Western analysis confirmed that the FpvA proteins were expressed and present in the OMs of cells with a mass of 85 kDa (Fig. (Fig.1C),1C), as found previously (21).

Alteration of the TonB box region was expected to abolish interactions between FpvA and TonB (and hence TonB-dependent functions of FpvA) without affecting other TonB-dependent functions of the cell. PAO fpvA attP::fpvAWT and PAO fpvA attP::fpvATBM were tested for growth in King's B broth medium (22) containing the iron-chelating compound EDDHA, which inhibits the growth of strains defective for pyoverdine-dependent iron uptake (2). PAO fpvA attP::fpvAWT had no significant growth deficiency compared to the wild-type strain PAO1, whereas PAO fpvA attP::fpvATBM showed no significant growth after 10 h, similar to the PAO fpvA mutant (Fig. (Fig.1D).1D). These data show that mutation of the TonB box prevented the ferripyoverdine transport function of the FpvA receptor, consistent with an inability of FpvATBM to interact with TonB.

Pyoverdine signaling is required for maximal expression of pyoverdine synthesis genes (25). Pyoverdine-mediated signaling was assessed by measuring PvdS-dependent gene expression using the reporter plasmid pMP190::PpvdE (40, 53). Signaling, represented by an increase in PvdS-dependent gene expression in response to added pyoverdine, was observed with bacteria carrying wild-type FpvA, but not with bacteria carrying FpvA with the TonB box mutation (Fig. (Fig.2).2). Furthermore, pyoverdine production was measured in P. aeruginosa strains. PAO fpvA attP::fpvATBM (expressing FpvA with the altered TonB box) produced an amount of pyoverdine similar to that produced by the PAO fpvA mutant and significantly less than PAO fpvA attP::fpvAWT or wild-type bacteria produced (Table (Table2).2). Together, these data are consistent with the hypothesis that FpvA-TonB interaction is required for pyoverdine-mediated signaling to occur.

FIG. 2.
Effect of TonB box mutation on pyoverdine-mediated signaling. PAO pvdF fpvA attP::fpvAWT (pMP190::PpvdE) and PAO pvdF fpvA attP::fpvATBM (pMP190::PpvdE) were grown in King's B medium, and β-galactosidase activity was assayed. Black bars, pyoverdine ...
TABLE 2.
Pyoverdine production by P. aeruginosa strains

We next investigated which of the P. aeruginosa TonB proteins was required for pyoverdine-mediated signaling. Signaling was assessed in P. aeruginosa tonB mutants using the pMP190::PpvdE reporter construct (Fig. (Fig.3).3). Mutation of tonB1 eliminated pyoverdine-mediated signaling. In contrast, mutations in tonB2, tonB3, or tonB2 and tonB3 together did not affect signaling. Assays in which the unrelated siderophore desferrioxamine was added in place of pyoverdine were also performed. No increase in β-galactosidase production was observed in response to desferrioxamine, indicating that the signaling response is specific for pyoverdine (Fig. (Fig.3).3). Strain PAO6609 and its derivatives yielded approximately threefold more enzyme units than derivatives of PAO pvdF (Fig. (Fig.2),2), but the reason for this is not known.

FIG. 3.
Pyoverdine-mediated signaling in TonB mutants. Strains were grown in King's B medium, and β-galactosidase activity was assayed. Black bars, pyoverdine was added; hashed bar, desferrioxamine was added. The results are averages of data from at least ...

Proteolysis of PvdS occurs in the absence of pyoverdine-mediated signaling (49). We therefore tested whether proteolysis of PvdS would occur in strains with the altered TonB box or in the tonB1 mutants. Proteolysis of PvdS did indeed occur in strains in which pyoverdine-mediated signaling did not take place (the TonB box mutant and tonB1 derivatives), even in the presence of pyoverdine, resulting in the occurrence of a subfragment of PvdS (Fig. (Fig.4).4). PvdS proteolysis was suppressed by the presence of pyoverdine in TonB1+ strains, including the tonB2 tonB3 double mutant.

FIG. 4.
Proteolysis of PvdS in the absence of pyoverdine-mediated signaling. Western analysis of whole-cell lysates from cultures grown to late exponential phase was carried out with an anti-PvdS monoclonal antibody (mAbI308). Lanes 1 and 2, PAO pvdF fpvA attP ...

Finally, to assess the possible involvement of tonB3 in iron uptake, growth assays were performed as described by Zhao and Poole (55). Unlike the tonB1 mutant, the pyoverdine-deficient PAO6609 tonB3 mutant showed no growth deficiency in iron-deficient succinate minimal medium compared to PAO6609 TonB+ (Fig. (Fig.5),5), indicating that TonB3 is not required for growth under iron limitation.

FIG. 5.
Growth of tonB mutants in succinate minimal medium. Strains: PAO6609 ([filled lozenge]); K1040 (tonB1) ([filled square]); K1407 (tonB2) ([filled triangle]); MS231 (tonB3) (×). The data are averages of three independent experiments.

Collectively these data show that TonB function is required for pyoverdine-mediated signaling, and of the three TonB proteins in P. aeruginosa, only TonB1 is able to perform this function. TonB1 is also the only TonB protein that enables uptake of ferripyoverdine (36). It has recently been proposed (44) that, following binding of ferripyoverdine by FpvA, interaction of TonB with the TonB box is involved in displacement of the signaling domain of FpvA, which then interacts with FpvR to trigger the signaling pathway. The requirement of TonB1 for both transport and signaling is consistent with this proposal, as is the effect of the FpvA TonB box mutation on both functions of FpvA. Expression of tonB2 is iron regulated, but TonB2 is not required for growth under iron limitation (55) or for signaling in the pyoverdine pathway, and no role has yet been assigned to TonB2. TonB3 is required for twitching motility and assembly of type IV pili (20), but TonB3, like TonB2, is not required for growth under iron limitation.

Bioinformatics analysis of completed genome sequences suggests that up to 30% of gram-negative bacteria possess more than one tonB gene (9). Among other bacterial species with multiple TonB proteins, there are several examples in which different TonB proteins have distinct functions in iron acquisition. For example, both redundant and specific functions have been reported for separate TonB proteins in S. marcescens (34). In this species, either TonB or the TonB-like protein HasB can mediate heme utilization. However TonB, but not HasB, is required for ferrisiderophore (enterochelin, ferrichrome, or ferric citrate) utilization. Similarly, in Vibrio cholerae, either of two TonB proteins can support growth with hemin, vibriobactin, or ferrichrome as the sole source of iron (32), but only TonB1 supports schizokinen utilization and only TonB2 supports enterobactin utilization (45).

Besides their roles in iron import, it is apparent that TonB proteins have important roles in signaling pathways. There are 35 predicted TonB-dependent receptors in P. aeruginosa PAO1, and an unusually high proportion of these (40%) are believed to encode TonB-dependent transducers (23). The occurrence of multiple TonB proteins in PAO1 may reflect an important regulatory role for these proteins, including involvement in siderophore-mediated signaling networks. The data presented here show that TonB1 is an important component of the pyoverdine-mediated signaling pathway in P. aeruginosa. It remains to be determined whether TonB2 and TonB3 play roles in iron acquisition or gene expression mediated by other siderophores.

Acknowledgments

This work was supported by a grant from the Royal Society of New Zealand Marsden Fund.

We gratefully acknowledge Keith Poole, who provided us with tonB mutant strains and with plasmid pJSS2 for deleting the genomic copy of fpvA; Karla Mettrick, who made the PAO fpvA and PAO pvdF fpvA strains; and Catherine Day, who provided advice throughout the course of this research.

Footnotes

[down-pointing small open triangle]Published ahead of print on 10 July 2009.

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