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.     

Siderophore cross-utilization amongst nodule isolates of the cowpea miscellany group and its effect on plant growth in the presence of antagonistic organisms

Nodule isolates from the cowpea miscellany group of legumes produced varying concentrations of catecholate and hydroxamate types of siderophores under iron-limiting conditions. The nodule isolates differed with respect to siderophore cross-utilizing abilities; some were proficient at using siderophores of other nodule isolates (homologous siderophores) while others could utilize siderophores produced by other rhizospheric bacteria (heterologous siderophores). Utilization of siderophore of rhizospheric bacterium PsB, a plant pathogen, benefited the nodule isolate G11 in terms of growth under iron-limiting laboratory conditions, while PsB was clearly inhibited in the presence of G11. Plate assays showed that siderophore of G11 could withhold iron from PsB and hence PsB was inhibited in the presence of G11. Isolates G11 and PsB when applied simultaneously to peanut seedlings under sterile soil conditions, provided a clear advantage to the plant in terms of reduction in the inhibitory effect of PsB. The count of the nodule isolate G11 increased in the soil when co-inoculated with PsB, as compared to when inoculated alone. Thus, the increased growth of the plant can be attributed to the iron sequestration and plant growth promoting properties of G11. The isolate G11 could utilize the siderophores produced by many other rhizospheric isolates while the siderophore of G11 was not being utilized by these rhizospheric isolates.     

Growth of Actinobacillus pleuropneumoniae is promoted by exogenous hydroxamate and catechol siderophores.

Siderophores bind ferric ions and are involved in receptor-specific iron transport into bacteria. Six types of siderophores were tested against strains representing the 12 different serotypes of Actinobacillus pleuropneumoniae. Ferrichrome and bis-catechol-based siderophores showed strong growth-promoting activities for A. pleuropneumoniae in a disk diffusion assay. Most strains of A. pleuropneumoniae tested were able to use ferrichrome (21 of 22 or 95%), ferrichrome A (20 of 22 or 90%), and lysine-based bis-catechol (20 of 22 or 90%), while growth of 36% (8 of 22) was promoted by a synthetic hydroxamate, N5-acetyl-N5-hydroxy-L-ornithine tripeptide. A. pleuropneumoniae serotype 1 (strain FMV 87-682) and serotype 5 (strain 2245) exhibited a distinct yellow halo around colonies on Chrome Azurol S agar plates, suggesting that both strains can produce an iron chelator (siderophore) in response to iron stress. The siderophore was found to be neither a phenolate nor a hydroxamate by the chemical tests of Arnow and Csaky, respectively. This is the first report demonstrating the production of an iron chelator and the use of exogenous siderophores by A. pleuropneumoniae. A spermidine-based bis-catechol siderophore conjugated to a carbacephalosporin was shown to inhibit growth of A. pleuropneumoniae. A siderophore-antibiotic-resistant strain was isolated and shown to have lost the ability to use ferrichrome, synthetic hydroxamate, or catechol-based siderophores when grown under conditions of iron restriction. This observation indicated that a common iron uptake pathway, or a common intermediate, for hydroxamate- and catechol-based siderophores may exist in A. pleuropneumoniae.     

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.     

Siderophore production by Salmonella typhi

This study was initiated to determine the mechanism of iron-uptake in Salmonella typhi. When stressed for iron, microorganisms produce siderophores to obtain the necessary nutrient. Generally two types of siderophores exist: the phenolate-type predominantly produced by bacteria and the hydroxamate-type commonly secreted by fungi. Results of this investigation showed that S. typhi produced siderophores of the phenolate-type since culture supernatant of the organism grown under iron-deprivation supported the growth of the phenolate-dependent auxotroph. The culture supernatant when extracted for phenolate siderophores, also supported the growth of the phenolate auxotroph but not the hydroxamate auxotroph. Production of phenolate-type siderophores were further confirmed using biochemical assays. These results showed that S. typhi utilized the high-affinity iron transport system to obtain the necessary iron.     

Utilization of siderophores by Candida albicans

Candida albicans secretes both hydroxamate and phenolate-type siderophores when grown under iron-restricted conditions. The inhibition of candidal growth by iron limitation was reversed by the addition of supplemental hydroxamate on phenolate siderophores. Both siderophores produced equal stimulation of growth suggesting that C. albicans could utilize both siderophores with equal efficiency. Addition of heterologous siderophores from both bacteria and fungi also supported growth of the yeast in a deferrated medium. These results suggest that C. albicans has an iron-uptake mechanism which enables it to obtain iron by utilizing candidal and non-candidal siderophores.     

Absence of siderophore activity in Legionella species grown in iron-deficient media

Growth of Legionella species in a defined medium deficient in iron did not result in the production of phenolic or hydroxamate siderophores which could be detected by chemical or biological assay methods. Growth of a variety of other gram-negative organisms under the same conditions resulted in the production of both hydroxamate and phenolate siderophores. The iron-deficient medium limited growth of the Legionella species more severely than it did the growth of the other gram-negative organisms. We have concluded that Legionella species do not make the commonly recognized siderophores, probably because they are restricted in their growth to those environments in which inorganic iron is readily available or is supplied in a form bound to an unknown carrier.     

Selective growth promotion and growth inhibition of Gram-negative and Gram-positive bacteria by synthetic siderophore-β-lactam conjugates

Conjugates of a carbacephalosporin with hydroxamate, spermexatol, N,N-bis(2,3-dihydroxybenzoyl)-L-lysine, mixed catecholate/hydroxamate and cyanuric acid-based siderophores were investigated for their potential to promote growth of siderophore indicator strains of Gram-negative and Gram-positive bacteria under iron depleted conditions, for their antibacterial activity and for their ability to use iron transport path-ways to penetrate the Gram-negative bacterial outer membrane. The selective growth promotion of enter-obacterial and pseudomonas strains by hydroxamate, spermexatol and mixed catecholate-hydroxamate siderophore-based conjugates bearing a L- or D-amino acid spacer was correlated with TonB dependent uptake routes. The preferred outer membrane siderophore receptor used in Escherichia coli was found to be Fiu, followed by Cir. Antagonistic effects of siderophores administered with the conjugates to determine antibacterial activity confirmed the active transport of conjugates via siderophore receptors. All of the conjugates were still able to diffuse through the porin proteins OmpC and OmpF. Nevertheless, strong inhibition of E. coli and Pseudomones aeruginosa outer membrane mutants DC2 and K799/61 compared to the parent strains indicated inefficient penetrability of all types of conjugates tested. Mycobacterium smegmatis SG 987 was able to use all of the siderophore-cephalosporin conjugates as growth promotors. Consequently there was no growth inhibition of this strain. © Rapid Science 1998.     

Iron transport in Streptomyces pilosus mediated by ferrichrome siderophores, rhodotorulic acid, and enantio-rhodotorulic acid

Streptomyces pilosus is one of several microbes which produce ferrioxamine siderophores. In the accompanying paper (G. Müller and K. Raymond, J. Bacteriol. 160:304-312), the mechanism of iron uptake mediated by the endogenous ferrioxamines B, D1, D2, and E was examined. Here we report iron transport behavior in S. pilosus as mediated by the exogenous siderophores ferrichrome, ferrichrysin, rhodotorulic acid (RA), and synthetic enantio-RA. In each case iron acquisition depended on metabolic energy and had uptake rates comparable to that of [55Fe]ferrioxamine B. However, the synthetic ferric enantio-RA (which has the same preferred chirality at the metal center as ferrichrome) was twice as effective in supplying iron as was the natural ferric RA complex, suggesting that stereospecific recognition at the metal center is involved in the transport process. Iron uptake mediated by ferrichrome and ferric enantio-RA was strongly inhibited by kinetically inert chromic complexes of desferrioxamine B. These inhibition experiments indicate that iron from these exogenous siderophores is transported by the same uptake system as ferrioxamine B. Since the ligands have no structural similarity to ferrioxamine B except for the presence of three hydoxamate groups, we conclude that only the hydroxamate iron center and its direct surroundings are important for recognition and uptake. This hypothesis is supported by the fact that ferrichrome A and ferrirubin, which are both substituted at the hydroxamate carbonyl groups, were not (or were poorly) effective in supplying iron to S. pilosus.     

Chemical Characterization,Crossfeeding and Uptake Studies on Hydroxamate Siderophore of <Emphasis Type="Italic">Alcaligenes faecalis</Emphasis>

We report the production of two types of siderophores namely catecholate and hydroxamate in modified succinic acid medium (SM) from Alcaligenes faecalis. Two fractions of siderophores were purified on amberlite XAD, major fraction was hydroxamate type having a λ_max at 224 nm and minor fraction appeared as catecholate with a λ_max of 264 nm. The recovery yield obtained from major and minor fractions was 297 and 50 mg ml⁻¹ respectively. The IEF pattern of XAD-4 purified siderophore suggested the pI value of 6.5. Cross feeding studies revealed that A. faecalis accepts heterologous as well as self (hydroxamate) siderophore in both free and iron complexed forms however; the rate of siderophore uptake was more in case of siderophores complexed to iron. Siderophore iron uptake studies indicated the differences between hydroxamate siderophore of A. faecalis and Alc E, a siderophore of Alcaligenes eutrophus.     

Bacterial siderophores efficiently provide iron to iron-starved tomato plants in hydroponics culture

Iron is one of the essential elements for a proper plant development. Providing plants with an accessible form of iron is crucial when it is scant or unavailable in soils. Chemical chelates are the only current alternative and are highly stable in soils, therefore, posing a threat to drinking water. The aim of this investigation was to quantify siderophores produced by two bacterial strains and to determine if these bacterial siderophores would palliate chlorotic symptoms of iron-starved tomato plants. For this purpose, siderophore production in MM9 medium by two selected bacterial strains was quantified, and the best was used for biological assay. Bacterial culture media free of bacteria (S) and with bacterial cells (BS), both supplemented with Fe were delivered to 12-week-old plants grown under iron starvation in hydroponic conditions; controls with full Hoagland solution, iron-free Hoagland solution and water were also conducted. Treatments were applied twice along the experiment, with a week in between. At harvest, plant yield, chlorophyll content and nutritional status in leaves were measured. Both the bacterial siderophore treatments significantly increased plant yield, chlorophyll and iron content over the positive controls with full Hoagland solution, indicating that siderophores are effective in providing Fe to the plant, either with or without the presence of bacteria. In summary, siderophores from strain Chryseobacterium C138 are effective in supplying Fe to iron-starved tomato plants by the roots, either with or without the presence of bacteria. Based on the amount of siderophores produced, an effective and economically feasible organic Fe chelator could be developed.     

Siderophore Cross-Utilization Amongst Rhizospheric Bacteria and the Role of Their Differential Affinities for Fe3+ on Growth Stimulation Under Iron-Limited Conditions

The majority of bacteria isolated from rhizospheres of Arachis hypogea (Groundnut) and Vigna radiata (Mung bean) predominantly produced catechol-type siderophores except for a few fluorescent pseudomonads that produced hydroxamates in addition to catecholates. The rhizospheric isolates differed in their ability to cross-utilize siderophores produced by other rhizospheric isolates (heterologous); some were highly proficient at utilizing heterologous siderophores, while others were poor cross-utilizers. Isolate G9, which utilized hydroxamate as well as catecholate siderophores, was found to be an efficient siderophore cross-utilizer, while isolates G2 and G6 were poor-utilizers of catecholate and non-utilizers of hydroxamate siderophores. Growth stimulation of two isolates G9 and G6 was seen when grown in the presence of externally supplied heterologous siderophores, which they cross-utilized. The iron-regulated outer membrane protein (IROMP) profiles differed for the most cross-utilizer and the least cross-utilizer strains, but in both the cases no new outer membrane proteins (OMP) were induced in response to the exogenous siderophores supplied. The growth of the organisms in the presence of heterologous siderophores that they failed to cross-utilize led to growth inhibition in the case of isolate G9. This appears to be due to a lower affinity of the siderophore of G9 as compared to the exogenously supplied G6 siderophore. A simple method was devised to measure relative affinities of respective siderophores for iron based on CAS solution decolorization by the siderophore preparations. The effect on the growth of the differential affinities of the siderophores for iron and the interactions of the organisms through cross-utilization is also discussed.     

Siderophore-mediated strategies of iron acquisition by extraintestinal isolates of Enterobacter spp

A total of 89 examined Enterobacter isolates belonging to three species: E. cloaceae, E. aerogenes and E. sakazakii, produced iron chelators detected in universal CAS assay. In chemical assays the strains were shown to excrete mostly catecholate (88 strains) and hydroxamate (42 strains) type of siderophores. Forty-one strains produced both catecholate and hydroxamate siderophores whereas one isolate produced only hydroxamate. Besides, the isolates were screened for genes coding for another siderophore: yersiniabactin. The genes for biosynthesis and uptake of yersiniabactin are located on the high-pathogenicity island (HPI) of Yersinia spp. The presence of three marker genes irp1, irp2 and fyuA was estimated by polymerase chain reaction. Two strains: E. aerogenes and E. cloaceae possessed irp1, irp2 and fyuA genes. PCR products of irp1, irp2 and fyuA were of 240, 280 and 780 bp, respectively.     

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.     

细菌铁载体拮抗植物病原真菌及促生作用研究进展

铁载体是微生物在缺铁条件下分泌的小分子有机化合物,以获取铁元素维持其生长。细菌分泌的铁载体在拮抗植物病原菌和促进植物生长方面具有重要作用。本文总结了细菌铁载体拮抗植物病原真菌的营养和生态位竞争、诱导植物诱导性系统抗性、扰乱病原菌铁稳态的机制,以及促进植物生长的作用,以解释细菌分泌的铁载体在多功能微生物菌剂研制中的重要作用。     

Growth ofNeisseria gonorrhoeae in media deficient in iron without detection of siderophores

A disseminating strain ofNeisseria gonorrhoeae was grown in a liquid, defined medium containing different levels of a strong chelator of ferric iron, ethylenediamine-di (o-hydroxyphenyl acetic acid) (EDDA). The inhibition of growth produced by EDDA was relieved by addition of iron. Filtrates from cultures grown in the media deficient in iron were examined for presence of siderophores, microbial chelators of iron, by colorimetric tests for phenolates and hydroxamates and by two biological assays in which the filtrates were tested for ability to stimulate growth of small inocula and for ability to prevent bactericidal effects of normal human serum. By use of these methods, none of the filtrates displayed evidence of gonococcal siderophores.     

Bacterial siderophores promote plant growth: Screening of catechol and hydroxamate siderophores

The aim of the study was to determine the quality and quantity of siderophores produced by bacteria isolated from plants' roots. The second aim was to determine the effect of siderophores on plants growth (Festuca rubra L. and Brassica napus L.). The study was carried out using bacteria isolated from roots of: Arabidopsis thaliana L., F. rubra, and Agrostis capillaris L., growing on the heavy metals contaminated area. The chrome azurol sulfonate (CAS) test, Arnow's test for catechol siderophores, and Csaksy's test for hydroxamate siderophores were performed. Among the bacteria, 42 isolates (39%) had a positive result in the CAS. Endophytic bacteria were mostly producing the catechol siderophores. It was found that F. rubra is the plant which is linked with the highest number of siderophores producing bacteria. The highest concentration of siderophores was noted for ectorhizospheric bacteria associated with A. thaliana, hyperaccumulating plant. It was found that hydroxamate siderophores are mainly produced by ectorhizosphere and rhizoplane bacteria. The siderophores producing bacteria reduced the toxicity of metals and improved the phytoremediation. Siderophores treatment increased the growth of plants in the biological assay, growing on two different soils: one highly contaminated with heavy metals and the second strongly alkaline soil.     

Siderophores of Cunninghamella blakesleeana NCIM 687

The fungus Cunninghamella blakesleeana NCIM 687, industrially recognized for progesterone biotransformation, was found to produce two siderophores at low stress of iron (upto 40 M iron in the growth medium). HPLC analysis and direct comparison with authentic samples characterized one of them as ferrichrysin (hydroxamate type) and other probably as a member of the coprogen family of siderophores.     

Molecular recognition of synthetic siderophore analogues: A study with receptor-deficient and fhu(A-B) deletion mutants of Escherichia coli

The biological activity of six synthetic siderophore analogues (two dihydroxamates, two trihydroxamates, one tetrahydroxamate and one 3-hydroxy-4(1H)pyridinone) has been studied in Escherichia coli, Morganella morganii 13 and Proteus mirabilis 8993 strains by using growth promotion tests. Various transport-deficient mutants of E. coli were used to study the route of entry into gram-negative bacteria. The results indicated that the synthetic hydroxamate compounds are transported via Fhu-mediated transport systems, although receptor specificity was low. This could be proven by using a delta (fhuA-B) E. coli mutant as a control in which growth promotion by natural hydroxamates was completely abolished, suggesting that a periplasmic binding-protein-dependent transport system (FhuB, C, D) is required for the transport of all synthetic ferric hydroxamate complexes. Although utilization of the synthetic hydroxamates was generally lower than that of the natural siderophores, differences in growth promotion could be detected. Highest activity was observed with the dihydroxamate DOCYDHAMA ligand which supported growth at concentrations <1 mM. In comparison with other polyamino-polyhydroxamate ligands studied, this dihydroxamate ligand has an extra diamide backbone that could be important for the interaction with the receptors or with FhuD. The synthetic trihydroxamate and tetrahydroxamate ligands showed a relatively low siderophore activity. Studies with Proteus and Morganella in the presence of increasing bipyridyl concentrations showed a decreased growth promotion with the synthetic ferric hydroxamates, suggesting the involvement of a reduction step during iron mobilization or an increased toxicity of bipyridyl. This was not observed in the case of the 3-hydroxy-4(1H)pyridinone where bipyridyl had no effect.     

Serpentine and non-serpentine Silene dioica plants do not differ in nickel tolerance

Serpentine soils are hostile to plant life. They are dry, contain high concentrations of nickel and have an unfavorable calcium/magnesium ratio. The dioecious plant Silene dioica (L.) Clairv. (Caryophyllaceae) is the most common herb on serpentine soils in the Swedish mountains. It also commonly grows on non-serpentine soils in the subalpine and coastal area. I have compared the germination frequency, plant establishment and growth of serpentine and subalpine non-serpentine populations in serpentine soil under greenhouse conditions. Further more I have studied the specific effect of nickel on root and shoot growth of serpentine and non-serpentine plants from the subalpine and coastal area in solutions with different concentrations of nickel. Plants from serpentine and non-serpentine populations grew well and in a similar fashion in serpentine soil. Moreover, S. dioica plants, irrespective of original habitat, tolerated enhanced concentrations of nickel when grown in solutions. An analysis of metal content in serpentine plants from natural populations shows that S. dioica has a higher nickel concentration in the roots than in the shoots. The growth studies show that S. dioica is constitutively adapted to serpentine, and that all populations have the genetic and ecological tolerance to grow on serpentine.