Iron Bound-Siderophores, Cyanic Acid, and Antibiotics Involved in Suppression of Thielaviopsis basicola by a Pseudomonas fluorescens Strain

A Pseudomonas fluorescens strain (CHAo) involved in suppression of black root rot caused by Thielaviopsis basicola in the field, inhibited T. basicola when colonizing roots grown under sterile conditions or when grown on culture, media. Under these conditions it produced siderophores (iron chelating compounds), cyanic acid, and several antibiotics. Iron-free siderophores inhibited neither the germination of endoconidia or chlamydospores nor the mycelial growth of T. basicola, but reduced the production of endoconidia. On the contrary, siderophores complexed with Fe³⁺ strongly inhibited mycelium growth and spore germination; free iron was less toxic than iron-bound siderophores. Therefore, contrary to what was believed to date, siderophores seem to be toxic not because they deplete iron but because they increase its concentration to the point where it becomes highly toxic. Cyanic acid and the antibiotics also inhibited the growth of T. basicola. Whetherall these compounds are involved in disease control in the soil remains, however, to be determined.     

Characterization of siderophores from Ustilago maydis

Two siderophores, ferrichrome and ferrichrome A, were found in cultures of Ustilago maydis (DC) Corda. Both siderophores were found intracellularly and extracellularly. Their authenticity was confirmed by thin layer chromatography, HPLC, UV-visible spectrometry, paper electrophoresis, amino acid analysis, NMR and fast atom bombardment mass spectroscopy. Regulation of siderophore production by iron was examined. Repression of biosynthesis of extracellular siderophores occurred at 10^–5 M iron. Ferrichrome was found intracellularly at all iron concentrations employed; in general, ferrichrome A was not found to be cell-associated.     

Identification of new members within suites of amphiphilic marine siderophores

Marine bacterial isolates Vibrio sp. HC0601C5 and Halomonas meridiana str. HC4321C1 were isolated off the coast of southern California and were found to produce an expanded suite of previously identified amphiphilic siderophores. Specifically two new members of the amphibactin family, amphibactins S and T, which have a C14:1 ω-7 fatty acid and a saturated C12 fatty acid, respectively, were produced by Vibrio sp. HC0601C5. These siderophores are produced in addition to a number of previously described amphibactins and are excreted into the culture supernatant. Two new members of the aquachelin family of siderophores, aquachelins I and J, which have an hydroxylated C12 fatty acid and a saturated C10 fatty acid, respectively, were produced by Halomonas meridiana str. HC4321C1. These four new siderophores are more hydrophilic than their previously reported relatives, aquachelins A–D and the amphibactin suite of 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.     

Response of the cyanobacterium,Oscillatoria tenuis,to low iron environments: the effect on growth rate and evidence for siderophore production

Cyanobacteria vary in their ability to grow in media contaning low amounts of biologically available iron. Some strains, such as Oscillatoria tenuis, are well adapted to thrive in low-iron environments. We investigated the mechanism of iron scavenging in O. tenuis and found that this cyanobacterium has a siderophore-mediated iron transport system that differs significantly from the traditional hydroxamate-siderophore transport system reported from other cyanobacteria. Unlike other cyanobacteria, this strain produces two types of siderophores, a hydroxamate-type siderophore and a catechol-type siderophore. Production of these two siderophores is expressed at two different iron levels in the medium, suggesting two different iron regulated uptake systems. We compared the production of each siderophore with the growth rate of the culture and found that the production of the catechol siderophore enhances the growth rate of the cyanobacterium, whereas the cells maintain lower than maximal growth rates when only the hydroxamate-type siderophore is being produced.Abbreviation EDDA ethylene diamine di-(o-hydroxyphenylacetic acid)     

Oxalic acid,versatile peroxidase secretion and chelating ability of <Emphasis Type="Italic">Bjerkandera fumosa</Emphasis> in rich and limited culture conditions

Efficient ligninolytic systems of wood-degrading fungi include not only oxidizing enzymes, but also low-molecular-weight effectors. The ability of Bjerkandera fumosa to secrete oxalic acid and versatile peroxidase (VP) in nitrogen-rich and nitrogen-limited media was studied. Higher activity of VP was determined in the nitrogen-limited media but greater concentration of oxalic acid was observed in the cultures of B. fumosa without nitrogen limitation. Ferric ions chelating ability of Bjerkandera fumosa studied in ferric ions limited media was correlated with the increased level of oxalic acid. The presence of hydroxamate-type siderophores in B. fumosa media were also detected. Oxalate decarboxylase was found to be responsible for regulation of oxalic acid concentration in the tested B. fumosa cultures.     

Plant growth promotion traits of phosphobacteria isolated from Puna,Argentina

The ability of soil microorganisms to solubilize phosphate is an important trait of plant growth-promoting bacteria leading to increased yields and smaller use of fertilizers. This study presents the isolation and characterization of phosphobacteria from Puna, northwestern Argentina and the ability to produce phosphate solubilization, alkaline phosphatase, siderophores, and indole acetic acid. The P-solubilizing activity was coincidental with a decrease in pH values of the tricalcium phosphate medium for all strains after 72 h of incubation. All the isolates showed the capacity to produce siderophores and indoles. Identification by 16S rDNA sequencing and phylogenetic analysis revealed that these strains belong to the genera Pantoea, Serratia, Enterobacter, and Pseudomonas. These isolates appear attractive for exploring their plant growth-promoting activity and potential field application.     

Azotobacter vinelandii gene clusters for two types of peptidic and catechol siderophores produced in response to molybdenum

Aim: To characterize the complementary production of two types of siderophores in Azotobacter vinelandii. Methods and Results: In an iron‐insufficient environment, nitrogen‐fixing A. vinelandii produces peptidic (azotobactin) and catechol siderophores for iron uptake to be used as a nitrogenase cofactor. Molybdenum, another nitrogenase cofactor, was also found to affect the production level of siderophores. Wild‐type cells excreted azotobactin into molybdenum‐supplemented and iron‐insufficient medium, although catechol siderophores predominate in molybdenum‐free environments. Two gene clusters were identified to be involved in the production of azotobactin and catechol siderophores through gene annotation and disruption. Azotobactin‐deficient mutant cells produced catechol siderophores under the molybdenum‐supplemented and iron‐insufficient conditions, whereas catechol siderophore–deficient mutant cells extracellularly secreted excess azotobactin under iron‐deficient condition independent of the concentration of molybdenum. This evidence suggests that a complementary siderophore production system exists in A. vinelandii. Conclusions: Molybdenum was found to regulate the production level of two types of siderophores. Azotobacter vinelandii cells are equipped with a complementary production system for nitrogen fixation in response to a limited quantity of metals. Significance and Impact of the Study: This is the first study identifying A. vinelandii gene clusters for the biosynthesis of two types of siderophores and clarifying the relationship between them.     

Isolation and purification of siderophores produced by cyanobacteria,Synechococcus sp. PCC 7942 andAnabaena variabilis ATCC 29413

New siderophores were isolated and purified from the spent growth medium of the cyanobacteriaSynechococcus sp. PCC 7942 (Anacystis nidulans R2) andAnabaena variabilis ATCC 29413 by solvent extraction and thin-layer chromatography. For each species the siderophore was released into the medium when the cells were grown at low iron concentrations and was not found in the medium of cells grown in iron-sufficient medium. Through a series of biological and chemical tests, combined with spectral analysis, the dihydroxamate nature of each siderophore was confirmed. The siderophores produced bySynechococcus sp. PCC 7942 andA. variabilis had distinct relative molecular masses of 310–313 Da and 520–525 Da, respectively. Neither of the two strains produced Arnow-positive extracellular organics, which indicate the excretion of extracellular catechol-type siderophores.     

High-performance liquid chromatography of siderophores from fungi

Summary A reversed-phase HPLC separation of iron(III) chelates of 16 representative fungal siderophores including ferrichromes, coprogens and triacetylfusarinine C was established in order to investigate siderophore production of fungi. For comparison purposes, the widely used bacterial siderophore ferrioxamine B was included. Culture filtrates of the fungiPenicillium resticulosum, Fusarium dimerum, Aspergillus fumigatus andNeurospora crassa were quantitatively analyzed for the presence of known and unknown siderophores after growth in low-iron culture media and adsorption on XAD-2 columns using this HPLC separation system. Photodiode array detection allowed the distinction between siderophores and non-siderophores. According to their ultraviolet/visible spectra, a further classification of the siderophores into four types due to the number of anhydromevalonic acid residues per molecule (0–3) was possible.     

Effect of pH,L-ornithine and L-proline on the hydroxamate siderophore production by Hymenoscyphus ericae,a typical ericoid mycorrhizal fungus

Since ericoid mycorrhizae become dominant in heathland plant communities on acid soils, we assessed the effect of pH on the hydroxamate siderophore production by a typical ericoid mycorrhizal fungus under pure culture conditions. In addition, we determined whether the supplementation of the nutrient medium with L-ornithine or L-proline as precursors for hydroxamate siderophores would enhance their biosynthesis. The results indicate that the hydroxamate siderophore production by Hymenoscyphus ericae has its optimum at pH 4.5 (between 3.5 and 5.5). L-ornithine rather than L-proline appears to favour the biosynthesis of hydroxamate siderophores.     

The DNA gyrase inhibitors,nalidixic acid and oxolinic acid,prevent iron-mediated repression of catechol siderophore synthesis inAzotobacter vinelandii

Summary Low concentrations of nalidixic acid and oxolinic acid that were just inhibitory toAzotobacter vinelandii growth promoted the production of the catechol siderophores azotochelin and aminochelin, in the presence of normally repressive concentrations of Fe³⁺. There was a limited effect on the pyoverdin siderophore, azotobactin, where low concentrations of Fe³⁺ were rendered less repressive, but the repression by higher concentrations of Fe³⁺ was normal. These drugs did not induce high-molecular-mass iron-repressible outer-membrane proteins and similar effects on the regulation of catechol siderophore synthesis were not produced by novobiocin, coumermycin, or ethidium bromide. The timing of nalidixic acid and Fe³⁺ addition to iron-limited cells was critical. Nalidixic acid had to be added before iron-repression of catechol siderophore synthesis and before the onset of iron-sufficient growth. Continued production of the catechol siderophores, however, was not due to interference with normal iron uptake. These data indicated that nalidixic acid prevented normal iron-repression of catechol siderophore synthesis but could not reverse iron repression once it had ocurred. The possible roles of DNA gyrase activity in the regulation of catechol siderophore synthesis is discussed.     

Siderophores of Marinobacter aquaeolei: petrobactin and its sulfonated derivatives

Siderophores are low molecular weight, high-affinity iron(III) ligands, produced by bacteria to solubilize and promote iron uptake under low iron conditions. Two prominent structural features characterize the majority of the marine siderophores discovered so far: (1) a predominance of suites of amphiphilic siderophores composed of an iron(III)-binding headgroup that is appended by one or two of a series of fatty acids and (2) a prevalence of siderophores that contain α-hydroxycarboxylic acid moieties (e.g., β-hydroxyaspartic acid or citric acid) which are photoreactive when coordinated to Fe(III). Variation of the fatty acid chain length affects the relative amphiphilicity within a suite of siderophores. Catecholate sulfonation is another structural variation that would affect the hydrophilicity of a siderophore. In addition to a review of the marine amphiphilic siderophores, we report the production of petrobactin disulfonate by Marinobacter aquaeolei VT8. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of siderophore-mediated iron transport inGeotrichum candidum,a non-siderophore producer

Summary Geotrichum candidum is capable of utilizing iron from hydroxamate siderophores of different structural classes. The relative rates of iron transport for ferrichrome, ferrichrysin, ferrioxamine B, fusigen, ferrichrome A, rhodotorulic acid, coprogen B, dimerium acid and ferrirhodin were 100%, 98%, 74%, 59%, 49%, 35%, 24%, 12% and 11% respectively. Ferrichrome, ferrichrysine and ferrichrome A inhibited [⁵⁹Fe]ferrioxamine-B-mediated iron transport by 71%, 68% and 28% respectively when added at equimolar concentrations to the radioactive complex. The inhibitory mechanism of [⁵⁹Fe]ferrioxamine B uptake by ferrichrome was non-competitive (K _i 2.4 M), suggesting that the two siderophores do not share a common transport system. Uptake of [⁵⁹Fe]ferrichrome, [⁵⁹Fe]rhodotorulic acid and [⁵⁹Fe]fusigen was unaffected by competition with the other two siderophores or with ferrioxamine B. Thus,G. candidum may possess independent transport systems for siderophores of different structural classes. The uptake rates of [¹⁴C]ferrioxamine B and⁶⁷Ga-desferrioxamine B were 30% and 60% respectively, as compared to [⁵⁹Fe]ferrioxamine B. The specific ferrous chelates, dipyridyl and ferrozine at 6 mM, caused 65% and 35% inhibition of [⁵⁹Fe]ferrioxamine uptake. From these results we conclude that, although about 70% of the iron is apparently removed from the complex by reduction prior to being transported across the cellular membrane, a significant portion of the chelated ligand may enter the cell intact. The former and latter mechanisms seem not to be mutually exclusive.     

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.     

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.     

The design,synthesis and study of siderophore-antibiotic conjugates Siderophore mediated drug transport

Summary The use of conjugates of microbial iron chelators (siderophores) and antibiotics for illicit transport of antibiotics into cells is a potentially powerful method for the rational design of therapeutic agents. The structural complexity of most natural siderophores has impeded progress in this area. Described here are the design, syntheses and preliminary biological studies of several siderophore--lactam antibiotic conjugates. Both hydroxamic-acid-based and catechol-based conjugates with and without amino acid spacers to carbacephalosporins were synthesized and demonstrated to be effective inhibitors ofEscherichia coli X580. Mutant selection was noted for each class of conjugates. Mutants selected from exposure of theE. coli to the hydroxamate conjugates were susceptible to the catechol conjugates and vice versa. Combinations of hydroxamate-and catechol-carbacephalosporin conjugates were most effective inhibitors ofE. coli X580.     

Accumulation of rare earth elements by siderophore-forming <Emphasis Type="Italic">Arthrobacter luteolus</Emphasis> isolated from rare earth environment of Chavara,India

In this study, Arthrobacter luteolus, isolated from rare earth environment of Chavara (Quilon district, Kerala, India), were found to produce catechol-type siderophores. The bacterial strain accumulated rare earth elements such as samarium and scandium. The siderophores may play a role in the accumulation of rare earth elements. Catecholate siderophore and low-molecular-weight organic acids were found to be present in experiments with Arthrobacter luteolus. The influence of siderophore on the accumulation of rare earth elements by bacteria has been extensively discussed.     

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.     

Occurrence of Strains Producing Specific Antibacterial Inhibitory Agents in Five Genera of Enterobacteriaceae

Striking differences in the production of specific inhibitory agents affecting other strains of the same (or of related) species were found between genera of the family Enterobacteriaceae. We tested 50–163 strains each of the potentially pathogenic genera: Escherichia, Citrobacter, Enterobacter, Kluyvera, and Leclercia for their ability to produce bacteriophages, high-molecular-weight (HMW) and low-molecular-weight (LMW) bacteriocins and siderophores against the same sets of strains, using the cross-test method. The genus Escherichia differs substantially from all other Enterobacteriaceae, harboring a notable proportion of lysogenic (36.6%) and colicinogenic (13.9%) strains. Only 18.2% of the Citrobacter strains are lysogenic and only rarely are they colicinogenic, although in 7.3%, they produce phage tail-like bacteriocins. On the other hand, Kluyvera strains were only in 1.8% lysogenic, no colicinogenic strains were found, but in 7.3%, they produced siderophores causing zones of growth inhibition in agar cultures of strains of the same genus. In Leclercia, 10.0% of the strains were lysogenic, 2.0% produced HMW bacteriocins, no colicinogenic strains were found and 2.0% produced siderophores. Enterobacter has shown 23.1% of strains producing siderophores, whereas merely 7.7% were lysogenic, 1.9% colicinogenic and 3.8% formed phage tail-like bacteriocins. HMW bacteriocins of Enterobacter strains disposed of an unusually wide spectrum of activity. The siderophore activity spectrum was rather wide in any genus, but the siderophores were usually not produced by strains producing phages or colicins.