Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Multiple outer membrane receptors for uptake of ferric pseudobactins inPseudomonas putida WCS358

Under iron limitationPseudomonas putida WCS358 produces a fluorescent siderophore, pseudobactin 358, which, after complexing iron, is transported back into the cell via the specific outer membrane receptor PupA. In addition, this strain has the capacity to take up iron via a large variety of siderophores produced by other fluorescent pseudomonads. Putative receptor genes for such siderophores were identified in the chromosome of strain WCS358 by PCR using primers matching two domains conserved in four ferric pseudobactin receptors, including PupA. Eleven amplification products within the expected size range were obtained. Sequence analysis confirmed that the products were derived from genes encoding outer membrane receptors. Two complete receptor genes were isolated from a genomic library ofP. putida WCS358. Both protein products are involved in the transport of a limited number of specific ferric pseudobactins. These results indicate that the ability ofP. putida WCS358 to exploit many different heterologous pseudobactins is related to the presence of multiple outer membrane receptor proteins.     

Cloning of the gene coding for the outer membrane receptor protein for ferric pseudobactin, a siderophore from a plant growth-promoting Pseudomonas strain

Plant growth-promoting Pseudomonas B10 produces its yellow-green, fluorescent siderophore (microbial iron transport agent) pseudobactin under iron-limiting conditions. A structural gene encoding the 85,000-Da putative outer membrane receptor protein for ferric pseudobactin was identified in a gene bank from Pseudomonas B10 prepared with the broad host-range conjugative cosmid cloning vector pLAFR1. Transposon Tn5 mutagenesis of recombinant plasmid pJLM300 localized the functional gene to a region of approximately 2.4 kilobases consistent with the apparent molecular weight of the receptor protein. Mobilization of pJLM300 into Pseudomonas A124 and A225, whose growth was inhibited by Pseudomonas B10 or pseudobactin, rendered these strains no longer susceptible to iron starvation by pseudobactin because they were now able to transport ferric pseudobactin. Pseudobactin biosynthetic genes flanked this receptor gene on both sides and were on separate operons. Transposon Tn5 insertion mutants of Pseudomonas B10 lacking this receptor protein were generated by a marker exchange technique and were defective in ferric pseudobactin transport. Such mutants could be complemented in trans by pJLM300. The production of pseudobactin, the receptor protein, and four other outer membrane proteins in Pseudomonas B10 was coordinately regulated by the level of intracellular iron.     

Characterization of Fluorescent Siderophore-Mediated Iron Uptake in Pseudomonas sp. Strain M114: Evidence for the Existence of an Additional Ferric Siderophore Receptor

In Pseudomonas sp. strain M114, the outer membrane receptor for ferric pseudobactin M114 was shown to transport ferric pseudobactins B10 and A225, in addition to its own. The gene encoding this receptor, which was previously cloned on pCUP3, was localized by Tn5 mutagenesis to a region comprising >1.6 kb of M114 DNA. A mutant (strain M114R1) lacking this receptor was then created by a marker exchange technique. Characterization of this mutant by using purified pseudobactin M114 in radiolabeled ferric iron uptake studies confirmed that it was completely unable to utilize this siderophore for acquisition of iron. In addition, it lacked an outer membrane protein band of 89 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, growth of the mutant was severely restricted under low-iron conditions. However, this phenotype was reversed in the presence of another fluorescent siderophore (pseudobactin MT3A) from Pseudomonas sp. strain MT3A, suggesting the presence of a second receptor in strain M114. Furthermore, wild-type Pseudomonas sp. strain B24 was not able to utilize ferric pseudobactin MT3A, and this phenotype was not reversed upon expression of the M114 receptor encoded on pCUP3. However, a cosmid clone (pMS1047) that enabled strain B24 to utilize ferric pseudobactin MT3A was isolated from an M114 gene bank. Radiolabel transport assays with purified pseudobactin MT3A confirmed this event. Plasmid pMS1047 was shown to encode an outer membrane protein of 81 kDa in strain B24 under iron-limiting conditions; this protein corresponds to a similar protein in strain M114.     

NMR assignments of the N-terminal signaling domain of the TonB-dependent outer membrane transducer PupB

Outer membrane TonB-dependent transducers (TBDTs) actively transport ferric siderophore complexes from the extracellular environment into Gram-negative bacteria. They also participate in a cell-surface signaling regulatory pathway that results in upregulation of the transducer itself, in response to iron-deplete conditions. The TBDT PupB transports ferric pseudobactin, and signals through its N-terminal signaling domain (NTSD), while the TBDT homolog PupA is signaling-inactive. Here, we report the NMR chemical shift assignments of the PupB-NTSD. This information will provide the basis for structural characterization of the PupB-NTSD to further explore its signaling properties.     

TonB or not TonB: is that the question?

Bacteria are able to survive in low-iron environments by sequestering this metal ion from iron-containing proteins and other biomolecules such as transferrin, lactoferrin, heme, hemoglobin, or other heme-containing proteins. In addition, many bacteria secrete specific low molecular weight iron chelators termed siderophores. These iron sources are transported into the Gram-negative bacterial cell through an outer membrane receptor, a periplasmic binding protein (PBP), and an inner membrane ATP-binding cassette (ABC) transporter. In different strains the outer membrane receptors can bind and transport ferric siderophores, heme, or Fe3+ as well as vitamin B12, nickel complexes, and carbohydrates. The energy that is required for the active transport of these substrates through the outer membrane receptor is provided by the TonB/ExbB/ExbD complex, which is located in the cytoplasmic membrane. In this minireview, we will briefly examine the three-dimensional structure of TonB and the current models for the mechanism of TonB-dependent energy transduction. Additionally, the role of TonB in colicin transport will be discussed.     

Iron transport-mediated antagonism between plant growth-promoting and plant-deleterious Pseudomonas strains

Both plant growth-promoting Pseudomonas B10 and its yellow-green, fluorescent iron transport agent (siderophore) pseudobactin enhance potato growth and biologically control certain soil-borne fungal diseases in part by depriving specific root-colonizing endemic microorganisms including phytopathogens of iron(III), thus inhibiting their growth. The present study examines this mode of iron deprivation. The growth inhibition of certain bean-deleterious fluorescent pseudomonads by specific bean-beneficial fluorescent pseudomonads is due in part to the inability of susceptible strains to utilize siderophores from beneficial strains to transport iron(III). Conversely, deleterious strains which were able to utilize siderophores from beneficial strains were not inhibited. The ability of a given pseudomonad to utilize another pseudomonad's siderophore may depend upon its possessing a specific outer membrane receptor protein for that pseudomonad's ferric siderophore. Siderophore-mediated competition for iron in microbial systems appears to be a widespread phenomenon.     

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.     

The ferric-pseudobactin receptor PupA of Pseudomonas putida WCS358: homology to TonB-dependent Escherichia coli receptors and specificity of the protein

The initial step in the uptake of iron via ferric pseudobactin by the plant-growth-promoting Pseudomonas putida strain WCS358 is binding to a specific outer-membrane protein. The nucleotide sequence of the pupA structural gene, which codes for a ferric pseudobactin receptor, was determined. It contains a single open reading frame which potentially encodes a polypeptide of 819 amino acids, including a putative N-terminal signal sequence of 47 amino acids. Significant homology, concentrated in four boxes, was found with the TonB-dependent receptor proteins of Escherichia coli. The pupA mutant MH100 showed a residual efficiency of 30% in the uptake of 55Fe3+ complexed to pseudobactin 358, whereas the iron uptake of four other pseudobactins was not reduced at all. Cells of strain WCS374 supplemented with the pupA gene of strain WCS358 could transport ferric pseudobactin 358 but showed no affinity for three other pseudobactins. It is concluded that PupA is a specific receptor for ferric pseudobactin 358, and that strain WCS358 produces at least one other receptor for other pseudobactins.     

Copurification of the FpvA ferric pyoverdin receptor of Pseudomonas aeruginosa with its iron-free ligand: implications for siderophore-mediated iron transport.

The Pseudomonas aeruginosa FpvA receptor is a TonB-dependent outer membrane transport protein that catalyzes uptake of ferric pyoverdin across the outer membrane. Surprisingly, FpvA expressed in P. aeruginosa grown in an iron-deficient medium copurifies with a ligand X that we have characterized by UV, fluorescence, and mass spectrometry as being iron-free pyoverdin (apo-PaA). PaA was absent from FpvA purified from a PaA-deficient P. aeruginosa strain. The properties of ligand binding in vitro revealed very similar affinities of apo-PaA and ferric-PaA to FpvA. Fluorescence resonance energy transfer was used to study in vitro the formation of the FpvA-PaA-Fe complex in the presence of PaA-Fe or citrate-Fe. The circular dichroism spectrum of FpvA indicated a 57% beta-structure content typical of porins and in agreement with the 3D structures of the siderophore receptors FhuA and FepA. In the absence of the protease's inhibitors, a truncated form of FpvA lacking 87 amino acids at its N-terminus was purified. This truncated form still bound PaA, and its beta-sheet content was conserved. This N-terminal region displays significant homology to the N-terminal periplasmic extensions of FecA from Escherichia coli and PupB from Pseudomonas putida, which were previously shown to be involved in signal transduction. This suggests a similar function for FpvA. The mechanism of iron transport in P. aeruginosa via the pyoverdin pathway is discussed in the light of all these new findings.     

Ferric iron transport system of Pseudomonas aeruginosa PAO1 that functions as the uptake pathway of a novel catechol-substituted cephalosporin,S-9096

A novel catechol-substituted cephalosporin, S-9096, showed potent antibacterial activity against Pseudomonas aeruginosa under both iron-deficient and aerobic conditions. S-9096 and ferric iron formed a chelate complex at the molar ratio of 3 to 1, which could be incorporated into P. aeruginosa cells grown under such conditions. Incorporation decreased when the cells were grown under either iron-sufficient or anaerobic conditions, with a concomitant disappearance of iron-regulated outer membrane proteins that were considered to function as receptors for ferric siderophores. These results indicated that the ferric chelate of S-9096 was incorporated into P. aeruginosa cells via a ferric iron transport pathway, which caused the high antibacterial potency of S-9096. All of the S-9096-resistant mutants that were able to grow even under iron-deficient conditions lacked an iron-regulated outer membrane protein having an apparent molecular mass of 66 kDa, suggesting the role of this protein as a receptor for the ferric chelate of S-9096. Correspondence to: Y. Yamano