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.     

Exchange of iron by gallium in siderophores

Siderophores are iron transport compounds produced by numerous microorganisms and which strongly chelate Fe(III), but not Fe(II). Other trivalent metals, such as Al(III), Cr(III), or Ga(III), are not capable of significantly displacing iron from siderophores. However, I demonstrate here that Ga(III) can effectively displace iron under reducing conditions. With ascorbate as reductant and ferrozine as Fe(II) trapping agent, the kinetics of reductive displacement of iron by Ga(III) were followed spectroscopically by the increase of absorbance at 562 nm due to formation of the Fe(II)-ferrozine complex. No significant reduction of siderophore occurred in the absence of Ga(III). With excess Ga(III), the displacement was quantitative and very rapid. The rate of metal exchange was pseudo first order with respect to Ga(III) concentration and highly pH dependent, suggesting that siderophore ligands are displaced from the iron in a concerted mechanism by Ga(III) and protonation to expose the Fe(III) to reduction by ascorbate. Reaction rates were dependent upon the structure of the siderophore, being greatest for ferric rhodotorulic acid and slowest for ferrichrome A at pH 5.4. The pH profile for ferric rhodotorulic acid was unusual in that it showed a maximum at pH 6.5, while all other siderophores examined showed an increase in rate as pH was lowered from 7.0. The physiological significance of this reaction to the clinical use of gallium is discussed.     

Utilization of microbial siderophores in iron acquisition by oat

Iron uptake by oat (Avena sativa cv Victory) was examined under hydroponic chemical conditions that required direct utilization of microbial siderophores for iron transport. Measurements of iron uptake rates by excised roots from the hydroxamate siderophores, ferrichrome, ferrichrome A, coprogen, ferrioxamine B (FOB), and rhodotorulic acid (RA) showed all five of the siderophores supplied iron, but that FOB and RA were preferentially utilized. FOB-mediated iron uptake increased four-fold when roots were preconditioned to iron stress and involved an active, iron-stress induced transport system that was inhibited by 5 millimolar sodium azide or 0.5 millimolar dinitrophenol. Kinetic studies indicated partial saturation with an apparent K_m of 5 micromolar when FOB was supplied at 0.1 to 50 micromolar concentrations. Whole plant experiments confirmed that 5 micromolar FOB was sufficient for plant growth. Siderophore-mediated iron transport was inhibited by Cr-ferrichrome, an analog of ferrated siderophore. Our results confirm the existence of a microbial siderophore iron transport system in oat which functions within the physiological concentrations produced and used by soil microorganisms.     

Mechanisms of iron acquisition from siderophores by microorganisms and plants

Most bacteria, fungi, and some plants respond to Fe stress by the induction of high-affinity Fe transport systems that utilize biosyrthetic chelates called siderophores. To competitively acquire Fe, some microbes have transport systems that enable them to use other siderophore types in addition to their own. Bacteria such as Escherichia coli achieve this ability by using a combination of separate siderophore receptors and transporters, whereas other microbial species, such as Streptomyces pilosus, use a low specificity, high-affinity transport system that recognizes more than one siderophore type. By either strategy, such versatility may provide an advantage under Fe-limiting conditions; allowing use of siderophores produced at another organism's expense, or Fe acquisition from siderophores that could otherwise sequester Fe in an unavailable form.Plants that use microbial siderophores may also be more Fe efficient by virtue of their ability to use a variety of Fe sources under different soil conditions. Results of our research examining Fe transport by oat indicate parity in plant and microbial requirements for Fe and suggest that siderophores produced by root-colonizing microbes may provide Fe to plants that can use the predominant siderophore types. In conjunction with transport mechanisms, ecological and soil chemical factors can influence the efficacy of siderophores and phytosiderophores. A model presented here attempts to incorporate these factors to predict conditions that may govern competition for Fe in the plant rhizosphere. Possibly such competition has been a factor in the evolution of broad transport capabilities for different siderophores by microorganisms and plants.     

Kinetics of iron acquisition from ferric siderophores by Paracoccus denitrificans.

The kinetics of iron accumulation by iron-starved Paracoccus denitrificans during the first 2 min of exposure to 55Fe-labeled ferric siderophore chelates is described. Iron is acquired from the ferric chelate of the natural siderophore L-parabactin in a process exhibiting biphastic kinetics by Lineweaver-Burk analysis. The kinetic data for 1 microM less than [Fe L-parabactin] less than 10 microM fit a regression line which suggests a low-affinity system (Km = 3.9 +/- 1.2 microM, Vmax = 494 pg-atoms of 55Fe min-1 mg of protein-1), whereas the data for 0.1 microM less than or equal to [Fe L-parabactin] less than or equal to 1 microM fit another line consistent with a high-affinity system (Km = 0.24 +/- 0.06 microM, Vmax = 108 pg-atoms of 55Fe min-1 mg of protein-1). The Km of the high-affinity uptake is comparable to the binding affinity we had previously reported for the purified ferric L-parabactin receptor protein in the outer membrane. In marked contrast, ferric D-parabactin data fit a single regression line corresponding to a simple Michaelis-Menten process with comparatively low affinity (Km = 3.1 +/- 0.9 microM, Vmax = 125 pg-atoms of 55Fe min-1 mg of protein-1). Other catecholamide siderophores with an intact oxazoline ring derived from L-threonine (L-homoparabactin, L-agrobactin, and L-vibriobactin) also exhibit biphasic kinetics with a high-affinity component similar to ferric L-parabactin. Circular dichroism confirmed that these ferric chelates, like ferric L-parabactin, exist as the lambda enantiomers. The A forms ferric parabactin (ferrin D- and L-parabactin A), in which the oxazoline ring is hydrolyzed to the open-chain threonyl structure, exhibit linear kinetics with a comparatively high Km (1.4 +/- 0.3 microM) and high Vmax (324 pg-atoms of 55Fe min-1 of protein-1). Furthermore, the marked stereospecificity seen between ferric D- and L-parabactins is absent; i.e., iron acquisition from ferric parabactin A is non stereospecific. The mechanistic implications of these findings in relation to a stereospecific high-affinity binding followed by a nonstereospecific postreceptor processing is discussed.     

The role of siderophores in iron acquisition by photosynthetic marine microorganisms

The photosynthetic picocyanobacteria and eukaryotic microorganisms that inhabit the open ocean must be able to supply iron for their photosynthetic and respiratory needs from the subnanomolar concentrations available in seawater. Neither group appears to produce siderophores, although some coastal cyanobacteria do. This is interpreted as an adaptation to the dilute oceanic environment rather than a phylogenetic constraint, since there are cases in which related taxa from different environments have the capacity to produce siderophores. Most photosynthetic marine microorganisms are presumably, however, capable of accessing iron from strong chelates since the majority of dissolved iron in seawater is complexed by organic ligands, including siderophores. Rather than direct internalization of siderophores and other iron chelates, marine organisms primarily appear to use uptake pathways that involve a reduction step to free bound iron, closely coupled with transport into the cell.     

Human iron transporters

Human iron transporters manage iron carefully because tissues need iron for critical functions, but too much iron increases the risk of reactive oxygen species. Iron acquisition occurs in the duodenum via divalent metal transporter (DMT1) and ferroportin. Iron trafficking depends largely on the transferrin cycle. Nevertheless, non-digestive tissues have a variety of other iron transporters that may render DMT1 modestly redundant, and DMT1 levels exceed those needed for the just-mentioned tasks. This review begins to consider why and also describes advances after 2008 that begin to address this challenge.     

Comparative structural analysis of TonB-dependent outer membrane transporters: implications for the transport cycle

TonB-dependent outer membrane transporters (TBDTs) transport organometallic substrates across the outer membranes of Gram-negative bacteria. Currently, structures of four different TBDTs have been determined by X-ray crystallography. TBDT structures consist of a 22-stranded beta-barrel enclosing a hatch domain. Structure-based sequence alignment of these four TBDTs indicates the presence of highly conserved motifs in both the hatch and barrel domains. The conserved motifs of the two domains are always in close proximity to each other and interact. We analyzed the very large interfaces between the barrel and hatch domains of TBDTs and compared their properties to those of other protein-protein interfaces. These interfaces are extensively hydrated. Most of the interfacial waters form hydrogen bonds to either the barrel or the hatch domain, with the remainder functioning as bridging waters in the interface. The hatch/barrel interfacial properties most resemble those of obligate transient protein complexes, suggesting that the interface is conducive to conformational change and/or movement of the hatch within the barrel. These results indicate that TBDTs can readily accommodate substantial conformational change and movement of their hatch domains during the active transport cycle. Also, these structural changes may require only modest forces exerted by the energy-coupling TonB protein upon the transporter.     

TonB-dependent iron acquisition: mechanisms of siderophore-mediated active transport†

Cells growing in aerobic environments have developed intricate strategies to overcome the scarcity of iron, an essential nutrient. In Gram-negative bacteria, high-affinity iron acquisition requires outer membrane-localized proteins that bind iron chelates at the cell surface and promote their uptake. Transport of bound chelates across the outer membrane depends upon TonB–ExbB–ExbD, a cytoplasmic membrane-localized complex that transduces energy from the proton motive force to high-affinity receptors in the outer membrane. Upon ligand binding to iron chelate receptors, conformational changes are induced, some of which are detected in the periplasm. These structural alterations signal the ligand-loaded status of the receptor and, therefore, the requirement for TonB-dependent energy transduction. Thus, TonB interacts preferentially and directly with ligand-loaded receptors. Such a mechanism ensures the productive use of cellular energy to drive active transport at the outer membrane.     

Coupled biogeochemical cycling of iron and manganese as mediated by microbial siderophores

Siderophores, biogenic chelating agents that facilitate Fe(III) uptake through the formation of strong complexes, also form strong complexes with Mn(III) and exhibit high reactivity with Mn (hydr)oxides, suggesting a pathway by which Mn may disrupt Fe uptake. In this review, we evaluate the major biogeochemical mechanisms by which Fe and Mn may interact through reactions with microbial siderophores: competition for a limited pool of siderophores, sorption of siderophores and metal–siderophore complexes to mineral surfaces, and competitive metal-siderophore complex formation through parallel mineral dissolution pathways. This rich interweaving of chemical processes gives rise to an intricate tapestry of interactions, particularly in respect to the biogeochemical cycling of Fe and Mn in marine ecosystems.     

The use of microbial siderophores for foliar iron application studies

Experiments were conducted to assess the distribution of foliar applied Fe-containing compounds using microbial siderophores. Fe was measured in leaf fluid obtained by centrifugation according to a determination method based on Fe chelation by desferrioxamine E and HPLC separation on a reversed phase column. To avoid sample Fe contamination, treatments were only applied to a part of the leaf following a systematic and reproducible procedure and iron concentration was exclusively determined in fluid obtained from non-treated leaf surfaces. The increase in leaf fluid Fe concentration associated with the distribution of leaf applied Fe-siderophores, Fe–EDTA and FeSO₄ × 7H₂O was evaluated using Vicia faba L., Nicotiana tabacum L. and Citrus madurensis Lour. plants. The method proved useful to investigate the process of leaf Fe penetration and its distribution within the plant. Evidence of the penetration and distribution of leaf applied Fe-rhizoferrin, Fe-coprogen hydrolysis products and Fe-dimerum acid is presented in this study.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5m and was completely inhibited at an iron concentration of 10m. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

Interactions between iron availability, aluminium toxicity and fungal siderophores

The influence of iron, aluminium and of the combined application of both metals on microbial biomass and production of siderophores by three fungi (Aspergillus nidulans, Neurospora crassa and Hymenoscyphus ericae) were investigated. All three species showed a strong iron regulation and Al-sensitivity of siderophore biosynthesis although several differences remained species dependent. Inhibitory effects of Fe and Al on siderophore-production were additive and the higher binding capacity of siderophores towards iron could be compensated by a higher Al-availability. Although pH itself is also important for regulation of siderophore biosynthesis, an indirect effect of Al on siderophore production via an Al-induced pH decrease could be outlined. The toxic effects of Al resulting in a reduced biomass production were compensated by high Fe-availability, whereas the addition of DFAM, a bacterial siderophore, enhanced Al-toxicity.     

Iron binding to Azotobacter salinestris melanin,iron mobilization and uptake mediated by siderophores

Iron-sufficient Azotobacter salinestris cells bound large amounts of ⁵⁵Fe to cell-associated catechol melanin in an energy-independent manner. Iron was mobilized from the cell surface by citric acid and transported into the cell in a process that was inhibited by azide, carbonyl cyanide m-chlorophenyl-hydrazone (CCCP), KCl or RbCl, the latter two known to inhibit Na⁺-dependent activities in A. salinestris. Iron-limited cells produced a hydroxamate compound (HDX) which promoted ⁵⁵Fe-uptake into iron-limited cells in a two step process. Initial uptake was inhibited by azide or CCCP, but not by KCl, while subsequent uptake was blocked by all inhibitors. Citric acid also mediated energy-dependent ⁵⁵Fe-uptake in iron-limited cells, but initial iron-uptake was less sensitive to CCCP than HDX-mediated iron-uptake. The results show that melanin serves as an iron trap, probably to protect the cells from oxidative damage mediated by H₂O₂ and the Fenton reaction. A model for HDX siderophore-mediated iron-uptake is proposed which requires energy to concentrate iron in the periplasm and H⁺/Na⁺-dependent events to bring iron into the cell.     

Distribution and functions of TonB-dependent transporters in marine bacteria and environments: implications for dissolved organic matter utilization

Background

Bacteria play critical roles in marine nutrient cycles by incorporating and redistributing dissolved organic matter (DOM) and inorganic nutrients in the ocean. TonB-dependent transporter (TBDT) proteins allow Gram-negative bacteria to take up scarce resources from nutrient-limiting environments as well as siderophores, heme, vitamin B12, and recently identified carbohydrates. Thus, the characterization of TBDT distribution and functions is essential to better understand the contribution TBDT to DOM assimilation and its consequences on nutrient cycling in the environment.

Methodology/Principal Findings

This study presents the distribution of encoded known and putative TBDT proteins in the genomes of microorganisms and from the Global Ocean Survey data. Using a Lek clustering algorithm and substrate specificities, the TBDT sequences were mainly classified into the following three groups: (1) DOM transporters; (2) Siderophores/Vitamins transporters; and (3) Heme/Hemophores/Iron(heme)-binding protein transporters. Diverse TBDTs were found in the genomes of oligotroph Citromicrobium bathyomarinum JL354 and Citromicrobium sp JLT1363 and were highly expressed in the stationary phase of bacterial growth. The results show that the Gammaproteobacteria and the Cytophaga-Flavobacterium-Bacteroides (CFB) group bacteria accounted for the majority of the TBDT gene pool in marine surface waters.

Conclusions/Significance

The results of this study confirm the ecological importance of TBDTs in DOM assimilation for bacteria in marine environments owing to a wide range of substrate utilization potential in the ubiquitous Gammaproteobacteria and CFB group bacteria.     

Influence of iron depletion on growth and production of catechol siderophores by different Frankia strains

Nine strains of Frankia isolated from six Casuarinaceae (including four Casuarina sp., one Allocasuarina and one Gymnostoma) and one Elaeagnaceae (Hippophae¨ rhamnoides) were screened for growth and production of siderophores in an iron-deficient liquid medium. Siderophore production was detected only in four strains (Cj, G2, CH and G82) using the CAS and Arnow assays. Salicylates formed more than 90% and dihydroxybenzoates formed less than 10% of all catechol-type siderophores produced. Growth of the former strains was less affected by iron deficiency than that of strains Rif, Thr, URU, BR and RT which do not produce siderophores. Optimal siderophore production by strain Cj was noted when iron concentration reached 0.5μm and was completely inhibited at an iron concentration of 10μm. The kinetics of siderophore production by strain Cj showed that siderophore synthesis was detectable during the growth stationary phase. Growth of Cj (a siderophore-producing strain) and of RT (a non-siderophore-producing strain) differed when 2,2-dipyridyl or ethylene di(o-hydroxyphenyl) acetic acid (EDDHA) was added to the iron-deficient growth medium. Frankia strain RT was the most sensitive to the detrimental effect of both iron chelators.     

The pyoverdin receptor FpvA, a TonB-dependent receptor involved in iron uptake by Pseudomonas aeruginosa (review)

Iron is an important element, essential for the growth of almost all living cells. Because of the high insolubility of iron(III) in aerobic conditions, many gram-negative bacteria produce, under iron limitation, small iron-chelating compounds called siderophores, together with new outer-membrane proteins, which function as receptors for the ferrisiderophores. Pseudomonas aeruginosa, an important human opportunistic pathogen, produces at least three known siderophores when grown in iron-deficient conditions: pyochelin, salicylate and pyoverdin. This review focuses on pyoverdin and on the ability of FpvA to bind iron-free and ferric-PaA pyoverdin, in the light of recent information gained from biochemical and biophysical studies and of the recently solved 3D-structures of the related ferrichrome FhuA and enterobactin FepA receptors in Escherichia coli.     

Root-microbial effects on plant iron uptake from siderophores and phytosiderophores

Collaborative experiments were conducted to determine whether microbial populations associated with plant roots may artifactually affect the rates of Fe uptake and translocation from microbial siderophores and phytosiderophores. Results showed nonaxenic maize to have 2 to 34-fold higher Fe-uptake rates than axenically grown plants when supplied with 1 μM Fe as either the microbial siderophore, ferrioxamine B (FOB), or the barley phytosiderophore, epi-hydroxymugineic acid (HMA). In experiments with nonsterile plants, inoculation of maize or oat seedlings with soil microorganisms and amendment of the hydroponic nutrient solutions with sucrose resulted in an 8-fold increase in FOB-mediated Fe-uptake rates by Fe-stressed maize and a 150-fold increase in FOB iron uptake rates by Fe-stressed oat, but had no effect on iron uptake by Fe-sufficient plants. Conversely, Fe-stressed maize and oat plants supplied with HMA showed decreased uptake and translocation in response to microbial inoculation and sucrose amendment. The ability of root-associated microorganisms to affect Fe-uptake rates from siderophores and phytosiderophores, even in short-term uptake experiments, indicates that microorganisms can be an unpredictable confounding factor in experiments examining mechanisms for utilization of microbial siderophores or phytosiderophores under nonsterile conditions.