Role of TonB1 in Pyoverdine-Mediated Signaling inPseudomonas aeruginosa

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-hydroxyphenylacetic 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 TonBExbB-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). TonBdependent 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 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 s...

Deciphering Protein Dynamics of the Siderophore Pyoverdine Pathway in Pseudomonas aeruginosa

“…The inner membrane protein TonB, part of the TonB-ExbB-ExbD complex, plays a crucial role in ferri-siderophore uptake by energizing the outer membrane transporters [22], [49] (for a review, see [50]). In TonB-mCHERRY photobleaching experiments, only 10±5% of the fluorescence was recovered within 20 s, evidence that a large proportion of TonB proteins were immobile; the diffusion coefficient of the mobile fraction of TonB-mCHERRY was 0.06±0.03 µm 2 s −1 ( Figure 2C ).…”

“…The mechanism of channel formation leading to import of the substrate through the outer membrane also remains to be elucidated but simulations indicate the interaction between TonB and the TonB box would lead to the plug domain unfolding [21]. In the genome of P. aeruginosa , three tonB genes have been identified but only tonB1 was found to be involved in PVD-dependent iron uptake [22]. Following its import into the periplasm, the PVD-Fe complex is dissociated through an iron reduction process [23].…”

Ferric-Pyoverdine Recognition by Fpv Outer Membrane Proteins of Pseudomonas protegens Pf-5

“…S1 and S2 in the supplemental material). FpvU shares four of six residues with the well-characterized TonB box of FpvAI (8,12), whereas the TonB boxes of the other five Fpvs of Pf-5 share only one to three of the six residues (see Fig. S2 in the supplemental material).…”

“…Transport of ferricsiderophore complexes by TBDPs requires energy, which is provided by proton motive force via TonB-ExbB-ExbD complexes in the inner membrane (11). In FpvAI, a well-characterized Fpv in P. aeruginosa, a 6-amino-acid motif called the TonB box is required for the interaction with TonB (9,12). Most Fpvs also have an N-terminal signaling domain that interacts with a regulatory protein (anti-sigma factor), which controls the expression of an extracytoplasmic function (ECF) sigma factor (13).…”

Legionella pneumophila LbtU Acts as a Novel, TonB-Independent Receptor for the Legiobactin Siderophore

“…Some bacteria have more than one tonB or tonB-like gene, and in all cases, the connection between TonB-ExbBD and iron acquisition, when examined, has been clear (1,4,10,29,39,58,59,64,93,97,121,126,139). But recently, it has become apparent that some bacteria that contain an OM and IM, including L. pneumophila, and species of Chlamydia, Chlamydophila, Coxiella, Ehrlichia, Francisella, and Rickettsia do not have TonB-ExbBD (29,74).…”