Effect of exogenous siderophores on iron uptake activity of marine bacteria under iron-limited conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N'-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using (59)Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Siderophores mediate reduced and increased uptake of cadmium by Streptomyces tendae F4 and sunflower (Helianthus annuus), respectively

Aims: As a toxic metal, cadmium (Cd) affects microbial and plant metabolic processes, thereby potentially reducing the efficiency of microbe or plant‐mediated remediation of Cd‐polluted soil. The role of siderophores produced by Streptomyces tendae F4 in the uptake of Cd by bacteria and plant was investigated to gain insight into the influence of siderophores on Cd availability to micro‐organisms and plants. Methods and Results: The bacterium was cultured under siderophore‐inducing conditions in the presence of Cd. The kinetics of siderophore production and identification of the siderophores and their metal‐bound forms were performed using electrospray ionization mass spectrometry. Inductively coupled plasma spectroscopy was used to measure iron (Fe) and Cd contents in the bacterium and in sunflower plant grown in Cd‐amended soil. Siderophores significantly reduced the Cd uptake by the bacterium, while supplying it with iron. Bacterial culture filtrates containing three hydroxamate siderophores secreted by S. tendae F4 significantly promoted plant growth and enhanced uptake of Cd and Fe by the plant, relative to the control. Furthermore, application of siderophores caused slightly more Cd, but similar Fe uptake, compared with EDTA. Bioinoculation with Streptomyces caused a dramatic increase in plant Fe content, but resulted only in slight increase in plant Cd content. Conclusion: It is concluded that siderophores can help reduce toxic metal uptake in bacteria, while simultaneously facilitating the uptake of such metals by plants. Also, EDTA is not superior to hydroxamate siderophores in terms of metal solubilization for plant uptake. Significance and Impact of the Study: The study showed that microbial processes could indirectly influence the availability and amount of toxic metals taken up from the rhizosphere of plants. Furthermore, although EDTA is used for chelator‐enhanced phytoremediation, microbial siderophores would be ideal for this purpose.     

Effect of Exogenous Siderophores on Iron Uptake Activity of Marine Bacteria under Iron-Limited Conditions

More than 60% of species examined from a total of 421 strains of heterotrophic marine bacteria which were isolated from marine sponges and seawater were observed to have no detectable siderophore production even when Fe(III) was present in the culture medium at a concentration of 1.0 pM. The growth of one such non-siderophore-producing strain, alpha proteobacterium V0210, was stimulated under iron-limited conditions with the addition of an isolated exogenous siderophore, N,N′-bis (2,3-dihydroxybenzoyl)-O-serylserine from a Vibrio sp. Growth was also stimulated by the addition of three exogenous siderophore extracts from siderophore-producing bacteria. Radioisotope studies using ⁵⁹Fe showed that the iron uptake ability of V0210 increased only with the addition of exogenous siderophores. Biosynthesis of a hydroxamate siderophore by V0210 was shown by paper electrophoresis and chemical assays for the detection of hydroxamates and catechols. An 85-kDa iron-regulated outer membrane protein was induced only under iron-limited conditions in the presence of exogenous siderophores. This is the first report of bacterial iron uptake through an induced siderophore in response to exogenous siderophores. Our results suggest that siderophores are necessary signaling compounds for growth and for iron uptake by some non-siderophore-producing marine bacteria under iron-limited conditions.     

Siderophore-based detection of Fe(iii) and microbial pathogens

Siderophores are low-molecular-weight iron chelators that are produced and exported by bacteria, fungi and plants during periods of nutrient deprivation. The structures, biosynthetic logic, and coordination chemistry of these molecules have fascinated chemists for decades. Studies of such fundamental phenomena guide the use of siderophores and siderophore conjugates in a variety of medicinal applications that include iron-chelation therapies and drug delivery. Sensing applications constitute another important facet of siderophore-based technologies. The high affinities of siderophores for both ferric ions and siderophore receptors, proteins expressed on the cell surface that are required for ferric siderophore import, indicate that these small molecules may be employed for the selective capture of metal ions, proteins, and live bacteria. This minireview summaries progress in methods that utilize native bacterial and fungal siderophore scaffolds for the detection of Fe(iii) or microbial pathogens.     

Potential of Siderophore Production by Bacteria Isolated from Heavy Metal: Polluted and Rhizosphere Soils

Recently, heavy metals have been shown to have a stimulating effect on siderophore biosynthesis in various bacteria. In addition, several studies have found that siderophore production is greater in bacteria isolated from soil near plant roots. The aim of this study was to compare the production of siderophores by bacterial strains isolated from heavy metal-contaminated and uncontaminated soils. Chrome azurol sulphonate was used to detect siderophore secretion by several bacterial strains isolated from heavy metal-contaminated and rhizosphere-uncontaminated soils with both a qualitative disc diffusion method and a quantitative ultraviolet spectrophotometric method. Siderophore production by rhizosphere bacteria was significantly greater than by bacteria isolated from contaminated soil. The Pearson’s correlation test indicated a positive correlation between the amount of siderophore produced by bacteria isolated from the rhizosphere using the quantitative and qualitative detection methods and the amount of heavy metal in the soil. However, a significant negative correlation was observed between the amount of siderophore produced by bacteria isolated from heavy metal-contaminated soil and the amount of heavy metal (r value of ?0.775, P < 0.001).     

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.     

Plant growth-promoting bacteria that decrease heavy metal toxicity in plants

Kluyvera ascorbata SUD165 and a siderophore-overproducing mutant of this bacterium, K. ascorbata SUD165/26, were used to inoculate tomato, canola, and Indian mustard seeds which were then grown in soil for 25-42 days in the presence of either nickel, lead, or zinc. The parameters that were monitored included plant wet and dry weight, protein and chlorophyll content in the plant leaves, and concentration of heavy metal in the plant roots and shoots. As indicated by a decrease in the measured values of these parameters, in all instances, plant growth was inhibited by the presence of the added metal. Both bacterial strains were effective, although not always to a statistically significant extent, at relieving a portion of the growth inhibition caused by the metals. In most cases, the siderophore overproducing mutant K. ascorbata 165/26 exerted a more pronounced effect on plant growth than did the wild-type bacterium K. ascorbata SUD165. The data suggest that the ability of these bacteria to protect plants against the inhibitory effects of high concentrations of nickel, lead, and zinc is related to the bacteria providing the plants with sufficient iron.     

Hydroxamate-siderophore production and utilization by marine eubacteria

Siderophore (iron-binding chelator) production was examined in 30 strains of open ocean bacteria from the generaVibrio, Alteromonas, Alcaligenes, Pseudomonas, andPhotobacterium. The results showed that hydroxamate-type siderophore production was widely distributed in various marine species, except for isolates ofAlteromonas macleodii andV. nereis. In all cases, the ability to produce siderophores was under the control of iron levels in the medium and satisfied the iron requirements of the siderophore bioassay organism. On the basis of chemical assay and bacterial bioassays, none of the examined isolates produced phenolate-type siderophores. Several isolates produces siderophores that were neither hydroxamatenor phenolate-type siderophores. Some strains such asAlteromonas communis produce siderophores that could be used by many other isolates. In contrast, the siderophore produced byAlcaligenes venustus had little cross-strain utilization. These findings suggest that the ability to produce siderophores may be common to open ocean bacteria.     

Disruption of N-acyl homoserine lactone-mediated cell signaling and iron acquisition in epiphytic bacteria by leaf surface compounds

Since N-acyl homoserine lactones (AHLs) are key mediators of cell density-dependent regulation of traits involved in virulence and epiphytic fitness in gram-negative bacteria such as Pseudomonas syringae, a variety of plant species were examined to determine their production of leaf surface compounds that could interact with these signaling systems. Leaf washings of 17 of 52 plant species tested stimulated or inhibited AHL-dependent traits in at least one of the bacterial reporter strains used. The active compounds from most plants could be distinguished from known AHLs due to different patterns of mobility during C8 and C18 reverse-phase thin-layer chromatography (TLC) and normal-phase TLC compared to the patterns for authentic bacterial AHLs. All plant extracts were also tested to determine their abilities to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron. Leaf washings from 16 of the 52 plant species, as well as tannic acid solutions, stimulated pyoverdine synthesis in P. syringae in a high-iron medium. These preparations also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not the growth of the wild-type bacterium. The stimulation of siderophore production and the growth inhibition by plant extracts and purified tannins were both reversed by addition of ferric chloride to culture media, indicating that iron was made unavailable by the compounds released onto the leaf surface.     

Catecholate Siderophores Protect Bacteria from Pyochelin Toxicity

Background

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.

Methods and Principal Findings

Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.

Conclusions

We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.     

Siderophores sorbed on Ca-montmorillonite as an iron source for plants

Previous investigations have shown significant sorption of siderophores to the solid phase in soils, and clay surfaces in particular. The ability of plants to utilize Fe from this reservoir is therefore of great interest. This research focused on the ability of the hydroxamate siderophore ferrioxamine B (FOB) sorbed to Ca-montmorillonite – prevailing in soils – to supply Fe to peanuts (Arachis hypogeae L.). Remediation of Fe deficiency by the sorbed siderophore was found to be similar to that by the free (unsorbed) form. The concentration needed to achieve complete remediation of chlorosis was one order of magnitude higher than that of the optimal FeEDDHA [Fe-ethylenediamine-di(o-hydroxyphenylacetic acid)]. Using dialysis tubes, it was shown that Fe uptake from the sorbed siderophore is executed mainly via long-range pathways and does not require close proximity to the plant roots. It was hypothesized that the process involves chelating agents in solution, which transport the Fe from the immobilized siderophore and enable its uptake by the plant. Under calcareous conditions, the ability of the sorbed FOB to supply Fe was significantly impaired, probably as a result of inactivation of the bridging mechanism. Various possible shuttle compounds were examined. EDDHA was found to be a very efficient shuttle compound, which caused complete remediation of Fe deficiency, even under very harsh calcareous conditions. The findings support our hypothesis and imply the effectiveness of a ligand-exchange mechanism to strategy I plants (commonly attributed to strategy II plants). We suggest that the secretion of substances with chelating abilities, which is usually considered a less effective means of Fe acquisition mechanism, takes on more importance in this context.     

Mechanisms of microbially enhanced Fe acquisition in red clover (Trifolium pratense L.)

Soil microorganisms may play an important role in plant Fe uptake from soils with low Fe bioavailability, but there is little direct experimental evidence to date. We grew red clover, an Fe-efficient leguminous plant, in a calcareous soil to investigate the role of soil microbial activity in plant Fe uptake. Compared with plants grown in non-sterlie (NS) grown plants, growth and Fe content of the sterile(s) grown plants was significantly inhibited, but was improved by foliar application of Fe EDTA, indicating that soil microbial activity should play an important role in plant Fe acquisition. When soil solution was incubated with phenolic root exudates from Fe-deficient red clover, a few microbial species thrived while growth of the rest was inhibited, suggesting that the Fe-deficient (-Fe) root exudates selectively influenced the rhizosphere's microbial community. Eighty six per cent of the phenolic-tolerant microbes could produce siderophore [the Fe(III) chelator] under -Fe conditions, and 71% could secrete auxin-like compounds. Interestingly, the synthetic and microbial auxins (MAs) significantly enhanced the Ferric reduction system, suggesting that MAs, in addition to siderophores, are important to plant Fe uptake. Finally, plant growth and Fe uptake in sterilized soil were significantly increased by rhizobia inoculation. Root Fe-EDTA reductase activity in the -Fe plant was significantly enhanced by rhizobia infection, and the rhizobia could produce auxin but not siderophore under Fe-limiting conditions, suggesting that the contribution of nodulating rhizobia to plant Fe uptake can be at least partially attributed to stimulation of turbo reductase activity through nodule formation and auxin production in the rhizosphere. Based on these observations, we propose as a model that root exudates from -Fe plants selectively influence the rhizosphere microbial community, and the microbes in turn favour plant Fe acquisition by producing siderophores and auxins.     

Recent insights into iron import by bacteria

Bacteria are confronted with a low availability of iron owing to its insolubility in the Fe3+ form or its being bound to host proteins. The bacteria cope with the iron deficiency by using host heme or siderophores synthesized by themselves or other microbes. In contrast to most other nutrients, iron compounds are tightly bound to proteins at the cell surfaces, from which they are further translocated by highly specific proteins across the cell wall of gram-positive bacteria and the outer membrane of gram-negative bacteria. Once heme and iron siderophores arrive at the cytoplasmic membrane, they are taken up across the cytoplasmic membrane by ABC transporters. Here we present an outline of bacterial heme and iron siderophore transport exemplified by a few selected cases in which recent progress in the understanding of the transport mechanisms has been achieved.     

Rhizosphere interactions and siderophores

Certain plant growth-promoting pseudomonads inhibit deleterious and pathogenic rhizosphere bacteria and fungi by producing siderophores. Properties of a siderophore transport system which might provide a competitive advantage under iron stress conditions include ability to utilize other organisms' siderophores, higher Fe(III) stability constant, faster kinetics of dissolution of Fe(III) minerals, more efficient transport system, and resistance to degradation. In order to determine the concentration and localization of siderophores in the rhizosphere monoclonal antibodies (Mabs) to ferric pseudobactin, the siderophore of Pseudomonas putida B10, have been developed. Several Mabs cross reacted differently with various pseudobactins. A growth medium has been developed for the study for siderophore-mediated rhizosphere interactions in the laboratory.     

Fusarinines and dimerum acid, mono- and dihydroxamate siderophores from Penicillium chrysogenum, improve iron utilization by strategy I and strategy II plants

Cucumber, as a strategy I plant, and Maize as a strategy II plant, were cultivated in hydroponic culture in the presence of a ferrated siderophore mixture (1 M) from a culture of Penicillium chrysogenumisolated from soil. The siderophore mixture significantly improved the iron status of these plants as measured by chlorophyll concentration to the same degree as a 100-fold higher FeEDTA supply. Analysis of the siderophore mixture from P. chrysogenum by HPLC and electrospray mass spectrometry revealed that besides the trihydroxamates, coprogen and ferricrocin, large amounts of dimerum acid and fusarinines were present which represent precursor siderophores or breakdown products of coprogen. In order to prove the iron donor properties of dimerum acid and fusarinines for plants, purified coprogen was hydrolyzed with ammonia and the hydrolysis products consisting of dimerum acid and fusarinine were used for iron uptake by cucumber and maize. In short term experiments radioactive iron uptake and translocation rates were determined using ferrioxamine B, coprogen and hydrolysis products of coprogen. While the trihydroxamates revealed negligible or intermediate iron uptake rates by both plant species, the fungal siderophore mixture and the ammoniacal hydrolysis products of coprogen showed high iron uptake, suggesting that dimerum acid and fusarinines are very efficient iron sources for plants. Iron reduction assays using cucumber roots or ascorbic acid also showed that iron bound to hydrolysis products of coprogen was more easily reduced compared to iron bound to trihydroxamates. Ligand exchange studies with epi-hydroxymugineic acid and EDTA showed that iron was easily exchanged between coprogen hydrolysis products and phytosiderophores or EDTA. The results indicate that coprogen hydrolysis products are an excellent source for Fe nutrition of plants.     

Characterization of Plant-Growth-Promoting Effects and Concurrent Promotion of Heavy Metal Accumulation in the Tissues of the Plants Grown in The Polluted Soil by Burkholderia Strain LD-11

Plant-growth-promoting (PGP) bacteria especially with the resistance to multiple heavy metals are helpful to phytoremediation. Further development of PGP bacteria is very necessary because of the extreme diversity of plants, soils, and heavy metal pollution. A Burkholderia sp. strain, numbered LD-11, was isolated, which showed resistances to multiple heavy metals and antibiotics. It can produce indole-3-acetic acid, 1-aminocyclopropane-1-carboxylic acid deaminase and siderophores. Inoculation with the LD-11 improved germination of seeds of the investigated vegetable plants in the presence of Cu, promoted elongation of roots and hypocotyledonary axes, enhanced the dry weights of the plants grown in the soils polluted with Cu and/or Pb, and increased activity of the soil urease and the rhizobacteria diversity. Inoculation with the LD-11 significantly enhanced Cu and/or Pb accumulation especially in the roots of the plants grown in the polluted soils. Notably, LD-11 could produce siderophores in the presence of Cu. Conclusively, the PGP effects and concurrent heavy metal accumulation in the plant tissues results from combined effects of the above-mentioned multiple factors. Cu is an important element that represses production of the siderophore by the bacteria. Phytoremediation by synergistic use of the investigated plants and the bacterial strain LD-11 is a phytoextraction process.     

Influence of iron on growth and extracellular products of Acinetobacter baumannii

Iron is an important nutrient required by bacteria for optimal growth. Acquisition of iron from the host where iron is restricted is an important mediator of bacterial pathogenesis. In iron deplete chemically defined medium (CDM-Fe) growth of Acinetobacter baumannii was restricted as compared to iron replete medium (CDM + Fe). Bacteria developed four high molecular weight outer membrane proteins (OMPs) of 88, 84, 80 and 77 kDa in CDM-Fe medium which were absent in CDM + Fe medium, and are known iron regulated outer membrane proteins (IROMPs). A. baumannii secreted siderophores extracellularly into the medium which act as iron chelators which had been demonstrated in the supernatants of CDM-Fe media. The siderophore was of catechol type. This shows that A. baumannii under iron restricted conditions express IROMPs along with production of catechol type siderophore in order to acquire iron from the external milieu.     

Disruption of N-Αcyl Homoserine Lactone-Mediated Cell Signaling and Iron Acquisition in Epiphytic Bacteria by Leaf Surface Compounds

Since N-acyl homoserine lactones (AHLs) are key mediators of cell density-dependent regulation of traits involved in virulence and epiphytic fitness in gram-negative bacteria such as Pseudomonas syringae, a variety of plant species were examined to determine their production of leaf surface compounds that could interact with these signaling systems. Leaf washings of 17 of 52 plant species tested stimulated or inhibited AHL-dependent traits in at least one of the bacterial reporter strains used. The active compounds from most plants could be distinguished from known AHLs due to different patterns of mobility during C₈ and C₁₈ reverse-phase thin-layer chromatography (TLC) and normal-phase TLC compared to the patterns for authentic bacterial AHLs. All plant extracts were also tested to determine their abilities to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron. Leaf washings from 16 of the 52 plant species, as well as tannic acid solutions, stimulated pyoverdine synthesis in P. syringae in a high-iron medium. These preparations also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not the growth of the wild-type bacterium. The stimulation of siderophore production and the growth inhibition by plant extracts and purified tannins were both reversed by addition of ferric chloride to culture media, indicating that iron was made unavailable by the compounds released onto the leaf surface.