TonB-dependent transporters and their occurrence in cyanobacteria

Background  

Different iron transport systems evolved in Gram-negative bacteria during evolution. Most of the transport systems depend on outer membrane localized TonB-dependent transporters (TBDTs), a periplasma-facing TonB protein and a plasma membrane localized machinery (ExbBD). So far, iron chelators (siderophores), oligosaccharides and polypeptides have been identified as substrates of TBDTs. For iron transport, three uptake systems are defined: the lactoferrin/transferrin binding proteins, the porphyrin-dependent transporters and the siderophore-dependent transporters. However, for cyanobacteria almost nothing is known about possible TonB-dependent uptake systems for iron or other substrates.     

The Pseudomonas aeruginosa pirA gene encodes a second receptor for ferrienterobactin and synthetic catecholate analogues

Actively secreted iron chelating agents termed siderophores play an important role in the virulence and rhizosphere competence of fluorescent pseudomonads, including Pseudomonas aeruginosa which secretes a high affinity siderophore, pyoverdine, and the low affinity siderophore, pyochelin. Uptake of the iron-siderophore complexes is an active process that requires specific outer membrane located receptors, which are dependent of the inner membrane-associated protein TonB and two other inner membrane proteins, ExbB and ExbC. P. aeruginosa is also capable of using a remarkable variety of heterologous siderophores as sources of iron, apparently by expressing their cognate receptors. Illustrative of this feature are the 32 (of which 28 putative) siderophore receptor genes observed in the P. aeruginosa PAO1 genome. However, except for a few (pyoverdine, pyochelin, enterobactin), the vast majority of P. aeruginosa siderophore receptor genes still remain to be characterized. Ten synthetic iron chelators of catecholate type stimulated growth of a pyoverdine/pyochelin deficient P. aeruginosa PAO1 mutant under condition of severe iron limitation. Null mutants of the 32 putative TonB-dependent siderophore receptor encoding genes engineered in the same genetic background were screened for obvious deficiencies in uptake of the synthetic siderophores, but none showed decreased growth stimulation in the presence of the different siderophores. However, a double knock-out mutant of ferrienterobactin receptor encoding gene pfeA (PA 2688) and pirA (PA0931) failed to be stimulated by 4 of the tested synthetic catecholate siderophores whose chemical structures resemble enterobactin. Ferric-enterobactin also failed to stimulate growth of the double pfeA-pirA mutant although, like its synthetic analogues, it stimulated growth of the corresponding single mutants. Hence, we confirmed that pirA represents a second P. aeruginosa ferric-enterobactin receptor. The example of these two enterobactin receptors probably illustrates a more general phenomenon of siderophore receptor redundancy in P. aeruginosa.     

Production of Siderophores Increases Resistance to Fusaric Acid in Pseudomonas protegens Pf-5

Fusaric acid is produced by pathogenic fungi of the genus Fusarium, and is toxic to plants and rhizobacteria. Many fluorescent pseudomonads can prevent wilt diseases caused by these fungi. This study was undertaken to evaluate the effect of fusaric acid on P. protegens Pf-5 and elucidate the mechanisms that enable the bacterium to survive in the presence of the mycotoxin. The results confirm that fusaric acid negatively affects growth and motility of P. protegens. Moreover, a notable increase in secretion of the siderophore pyoverdine was observed when P. protegens was grown in the presence of fusaric acid. Concomitantly, levels of enzymes involved in the biosynthesis of pyoverdine and enantio-pyochelin, the second siderophore encoded by P. protegens, increased markedly. Moreover, while similar levels of resistance to fusaric acid were observed for P. protegens mutants unable to synthesize either pyoverdine or enanto-pyochelin and the wild type strain, a double mutant unable to synthesize both kinds of siderophores showed a dramatically reduced resistance to this compound. This reduced resistance was not observed when this mutant was grown under conditions of iron excess. Spectrophotometric titrations revealed that fusaric acid binds not only Fe²⁺ and Fe³⁺, but also Zn²⁺, Mn²⁺ and Cu²⁺, with high affinity. Our results demonstrate that iron sequestration accounts at least in part for the deleterious effect of the mycotoxin on P. protegens.     

Iron transporters in marine prokaryotic genomes and metagenomes

In the pelagic environment, iron is a scarce but essential micronutrient. The iron acquisition capabilities of selected marine bacteria have been investigated, but the recent proliferation of marine prokaryotic genomes and metagenomes offers a more comprehensive picture of microbial iron uptake pathways in the ocean. Searching these data sets, we were able to identify uptake mechanisms for Fe(3+), Fe(2+) and iron chelates (e.g. siderophore and haem iron complexes). Transport of iron chelates is accomplished by TonB-dependent transporters (TBDTs). After clustering the TBDTs from marine prokaryotic genomes, we identified TBDT clusters for the transport of hydroxamate and catecholate siderophore iron complexes and haem using gene neighbourhood analysis and co-clustering of TBDTs of known function. The genomes also contained two classes of siderophore biosynthesis genes: NRPS (non-ribosomal peptide synthase) genes and NIS (NRPS Independent Siderophore) genes. The most common iron transporters, in both the genomes and metagenomes, were Fe(3+) ABC transporters. Iron uptake-related TBDTs and siderophore biosynthesis genes were less common in pelagic marine metagenomes relative to the genomic data set, in part because Pelagibacter ubique and Prochlorococcus species, which almost entirely lacked these Fe uptake systems, dominate the metagenomes. Our results are largely consistent with current knowledge of iron speciation in the ocean, but suggest that in certain niches the ability to acquire siderophores and/or haem iron chelates is beneficial.     

Diversity of siderophore-mediated iron uptake systems in fluorescent pseudomonads: not only pyoverdines

Fluorescent pseudomonads are gamma-proteobacteria known for their capacity to colonize various ecological niches. This adaptability is reflected by their sophisticated and diverse iron uptake systems. The majority of fluorescent pseudomonads produce complex peptidic siderophores called pyoverdines or pseudobactins, which are very efficient iron scavengers. A tremendous variety of pyoverdines has been observed, each species producing a different pyoverdine. This variety can be used as an interesting tool to study the diversity and taxonomy of fluorescent pseudomonads. Other siderophores, including newly described ones, are also produced by pseudomonads, sometimes endowed with interesting properties in addition to iron scavenging, such as formation of complexes with other metals or antimicrobial activity. Factors other than iron limitation, and different regulatory proteins also seem to influence the production of siderophores in pseudomonads and are reviewed here as well. Another peculiarity of pseudomonads is their ability to use a large number of heterologous siderophores via different TonB-dependent receptors. A first genomic analysis of receptors in four different fluorescent pseudomonads suggests that their siderophore ligand repertoire is likely to overlap, and that not all receptors recognize siderophores as ligands.     

Structure–function relationships in the bifunctional ferrisiderophore FpvA receptor from Pseudomonas aeruginosa

FpvA is the primary outer membrane transporter required for iron acquisition via the siderophore pyoverdine (Pvd) in Pseudomonas aeruginosa. FpvA, like other ferrisiderophore transporters, consists of a membrane-spanning β-barrel occluded by a plug domain. The β-strands of the barrel are connected by large extracellular loops and periplasmic turns. Like some other TonB-dependent transporters, FpvA has a periplasmic domain involved in a signalling cascade that regulates expression of genes required for ferrisiderophore transport. Here, the structures of FpvA in different loading states are analysed in light of mutagenesis data. This analysis highlights the roles of different protein domains in Pvd-Fe uptake and the signalling cascade and reveals a strong correlation between Pvd-Fe transport and activation of the signalling cascade. It is likely that conclusions drawn for FpvA will be relevant to other TonB-dependent ferrisiderophore transport and signalling proteins.     

Fate of ferrisiderophores after import across bacterial outer membranes: different iron release strategies are observed in the cytoplasm or periplasm depending on the siderophore pathways

Siderophore production and utilization is one of the major strategies deployed by bacteria to get access to iron, a key nutrient for bacterial growth. The biological function of siderophores is to solubilize iron in the bacterial environment and to shuttle it back to the cytoplasm of the microorganisms. This uptake process for Gram-negative species involves TonB-dependent transporters for translocation across the outer membranes. In Escherichia coli and many other Gram-negative bacteria, ABC transporters associated with periplasmic binding proteins import ferrisiderophores across cytoplasmic membranes. Recent data reveal that in some siderophore pathways, this step can also be carried out by proton-motive force-dependent permeases, for example the ferrichrome and ferripyochelin pathways in Pseudomonas aeruginosa. Iron is then released from the siderophores in the bacterial cytoplasm by different enzymatic mechanisms depending on the nature of the siderophore. Another strategy has been reported for the pyoverdine pathway in P. aeruginosa: iron is released from the siderophore in the periplasm and only siderophore-free iron is transported into the cytoplasm by an ABC transporter having two atypical periplasmic binding proteins. This review presents recent findings concerning both ferrisiderophore and siderophore-free iron transport across bacterial cytoplasmic membranes and considers current knowledge about the mechanisms involved in iron release from siderophores.     

1H, 13C and 15N resonance assignments of the periplasmic signalling domain of HasR, a TonB-dependent outer membrane heme transporter

TonB-dependent transporters (TBDTs) are bacterial outer membrane proteins that internalize nutrients such as vitamin B12, metal complexes, heme, some carbohydrates, etc. In addition to their transport activity, several TBDTs are also involved in a signalling cascade from the cell surface into the cytoplasm, via their periplasmic signalling domain. Here we report the backbone and side chain resonance assignments of the signalling domain of HasR, a TonB-dependent outer membrane heme transporter from Serratia marcescens as a first step towards its structural study.     

Characterization of a pyoverdine-deficient mutant of Pseudomonas fluorescens impaired in the secretion of extracellular lipase

A mutant of Pseudomonas fluorescens strain B52 deficient in the synthesis of the fluorescent pigment, pyoverdine, was isolated. Absence of pyoverdine and other siderophores was confirmed by gel filtration, a specific siderophore assay, and inhibition studies with the iron chelator EDDA. Both parent and mutant synthesized additional outer membrane proteins in response to iron-limitation. Mutant cells cultured in the absence of iron(III) accumulated ⁵⁵Fe-labeled pyoverdine. The mutant produced extracellular proteinase normally on various media, but was deficient in lipase secretion. Growth of the mutant with partially-purified pyoverdine resulted in a 2.5-fold stimulation of lipase secretion. The mutant grew poorly in deferrated medium; however, the addition of iron(III) stimulated growth. Proteinase secretion in deferrated medium was stimulated over a narrow range of iron(III) concentration, while lipase secretion was only slightly affected. The data suggest that separate regulatory mechanisms exist for the control of proteinase and lipase secretion by iron(III).Contribution No. 768 from the Food Research Centre     

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-dependent receptors—structural perspectives

Plants, bacteria, fungi, and yeast utilize organic iron chelators (siderophores) to establish commensal and pathogenic relationships with hosts and to survive as free-living organisms. In Gram-negative bacteria, transport of siderophores into the periplasm is mediated by TonB-dependent receptors. A complex of three membrane-spanning proteins TonB, ExbB and ExbD couples the chemiosmotic potential of the cytoplasmic membrane with siderophore uptake across the outer membrane. The crystallographic structures of two TonB-dependent receptors (FhuA and FepA) have recently been determined. These outer membrane transporters show a novel fold consisting of two domains. A 22-stranded antiparallel β-barrel traverses the outer membrane and adjacent β-strands are connected by extracellular loops and periplasmic turns. Located inside the β-barrel is the plug domain, composed primarily of a mixed four-stranded β-sheet and a series of interspersed α-helices. Siderophore binding induces distinct local and allosteric transitions that establish the structural basis of signal transduction across the outer membrane and suggest a transport mechanism.     

细菌内依赖TonB的外膜铁转运体的研究进展

铁是细菌所必需的微量营养元素,但由于易被氧化溶解性低,生物体的利用率大大降低。细菌在进化过程中形成多种策略来吸收环境中低浓度的铁,不同类型铁的吸收通过外膜上依赖TonB的转运体(TonB-dependent transporters,TBDTs)完成,TBDTs结合不同形式的铁复合物,通过内膜上的TonB-ExbB-ExbD复合物提供能量完成转运,对其机制的研究一直是微生物基础生命活动研究中的热点问题。近年来新鉴定了一些TBDTs的结构,并对其功能和转运机制有了更深入的研究,对此进行了综述,不仅有助于进一步揭示细菌的铁转运机制,而且有助于寻找新的靶位点以开发新的治疗药物。     

The heterologous siderophores ferrioxamine B and ferrichrome activate signaling pathways in Pseudomonas aeruginosa

Pseudomonas aeruginosa secretes two siderophores, pyoverdine and pyochelin, under iron-limiting conditions. These siderophores are recognized at the cell surface by specific outer membrane receptors, also known as TonB-dependent receptors. In addition, this bacterium is also able to incorporate many heterologous siderophores of bacterial or fungal origin, which is reflected by the presence of 32 additional genes encoding putative TonB-dependent receptors. In this work, we have used a proteomic approach to identify the inducing conditions for P. aeruginosa TonB-dependent receptors. In total, 11 of these receptors could be discerned under various conditions. Two of them are only produced in the presence of the hydroxamate siderophores ferrioxamine B and ferrichrome. Regulation of their synthesis is affected by both iron and the presence of a cognate siderophore. Analysis of the P. aeruginosa genome showed that both receptor genes are located next to a regulatory locus encoding an extracytoplasmic function sigma factor and a transmembrane sensor. The involvement of this putative regulatory locus in the specific induction of the ferrioxamine B and ferrichrome receptors has been demonstrated. These results show that P. aeruginosa has evolved multiple specific regulatory systems to allow the regulation of TonB-dependent receptors.     

Exogenous siderophore-mediated iron uptake in Pseudomonas aeruginosa: possible involvement of porin OprF in iron translocation.

In addition to the two siderophores pyoverdine and pyochelin synthesized by Pseudomonas aeruginosa ATCC 15692 (strain PAO1), several siderophores produced by other bacteria or fungi, namely cepabactin, salicylic acid, desferriferrichrysin, desferriferricrocin, desferriferrioxamine B, desferriferrioxamine E and coprogen, were able to promote iron uptake with variable efficiencies into this bacterium. For most of these siderophores, these results were consistent with the growth stimulation produced by the same compounds in a plate bioassay. Desferriferrichrome A, enterobactin and desferriferrirubin, however, did not promote iron uptake, although enterobactin and desferriferrirubin stimulated bacterial growth. These paradoxical data are discussed in view of siderophore-inducible iron uptake systems, as demonstrated recently for enterobactin. Among the strains tested, including the wild-type PAO1, the pyoverdine-less mutant PAO6606 and the two porin-mutants P. aeruginosa H636 (oprF::omega) and P. aeruginosa H673 (oprD::Tn501), only for the porin-OprF mutant were fewer siderophores able to promote iron uptake compared to the other strains. Such results suggest that beside specific routes for iron uptake P. aeruginosa is also able to take up siderophore-liganded iron through OprF.     

Pseudomonas fluorescens Pirates both Ferrioxamine and Ferricoelichelin Siderophores from Streptomyces ambofaciens

Iron is essential in many biological processes. However, its bioavailability is reduced in aerobic environments, such as soil. To overcome this limitation, microorganisms have developed different strategies, such as iron chelation by siderophores. Some bacteria have even gained the ability to detect and utilize xenosiderophores, i.e., siderophores produced by other organisms. We illustrate an example of such an interaction between two soil bacteria, Pseudomonas fluorescens strain BBc6R8 and Streptomyces ambofaciens ATCC 23877, which produce the siderophores pyoverdine and enantiopyochelin and the siderophores desferrioxamines B and E and coelichelin, respectively. During pairwise cultures on iron-limiting agar medium, no induction of siderophore synthesis by P. fluorescens BBc6R8 was observed in the presence of S. ambofaciens ATCC 23877. Cocultures with a Streptomyces mutant strain that produced either coelichelin or desferrioxamines, as well as culture in a medium supplemented with desferrioxamine B, resulted in the absence of pyoverdine production; however, culture with a double mutant deficient in desferrioxamines and coelichelin production did not. This strongly suggests that P. fluorescens BBbc6R8 utilizes the ferrioxamines and ferricoelichelin produced by S. ambofaciens as xenosiderophores and therefore no longer activates the production of its own siderophores. A screening of a library of P. fluorescens BBc6R8 mutants highlighted the involvement of the TonB-dependent receptor FoxA in this process: the expression of foxA and genes involved in the regulation of its biosynthesis was induced in the presence of S. ambofaciens. In a competitive environment, such as soil, siderophore piracy could well be one of the driving forces that determine the outcome of microbial competition.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> siderophores in the sputum of patients with cystic fibrosis

The lungs of patients with cystic fibrosis become chronically infected with the bacterium Pseudomonas aeruginosa, which heralds progressive lung damage and a decline in health. Iron is a crucial micronutrient for bacteria and its acquisition is a key factor in infection. P. aeruginosa can acquire this element by secreting pyoverdine and pyochelin, iron-chelating compounds (siderophores) that scavenge iron and deliver it to the bacteria. Siderophore-mediated iron uptake is generally considered a key factor in the ability of P. aeruginosa to cause infection. We have investigated the amounts of pyoverdine in 148 sputum samples from 36 cystic fibrosis patients (30 infected with P. aeruginosa and 6 as negative controls). Pyoverdine was present in 93 samples in concentrations between 0.30 and 51 μM (median 4.6 μM) and there was a strong association between the amount of pyoverdine and the number of P. aeruginosa present. However, pyoverdine was not present, or below the limits of detection (~0.3 μM), in 21 sputum samples that contained P. aeruginosa. Pyochelin was also absent, or below the limits of detection (~1 μM), in samples from P. aeruginosa-infected patients with little or no detectable pyoverdine. Our data show that pyoverdine is an important iron-scavenging molecule for P. aeruginosa in many cystic fibrosis patients, but other P. aeruginosa iron-uptake systems must be active in some patients to satisfy the bacterial need for iron.