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.     

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.     

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.     

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.     

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.     

Effects of iron level on the anatagonistic action of siderophores from non-pathogenicStaphylococcus spp

The production, detection and the effects of iron concentration on the siderophore production ofStaphylococcus strains used as meat starter cultures were studied. Non-pathogenicStaphylococcus strains produce extracellular low molecular weight compounds which exhibited positive reactivity when measured by a universal detection method for siderophores. The production of siderophores was very closely associated with the iron concentration in the medium, and very low additions considerably reduced siderophore production. Although the production of siderophores was highly iron-dependent, the antimicrobial activity of spent medium fromStaphylococcus cultures against selected yeasts and moulds remained considerable under high iron concentrations.     

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.     

Genomic insights into the iron uptake mechanisms of the biomining microorganism Acidithiobacillus ferrooxidans

Commercial bioleaching of copper and the biooxidation of gold is a cost-effective and environmentally friendly process for metal recovery. A partial genome sequence of the acidophilic, bioleaching bacterium Acidithiobacillus ferrooxidans is available from two public sources. This information has been used to build preliminary models that describe how this microorganism confronts unusually high iron loads in the extremely acidic conditions (pH 2) found in natural environments and in bioleaching operations. A. ferrooxidans contains candidate genes for iron uptake, sensing, storage, and regulation of iron homeostasis. Predicted proteins exhibit significant amino acid similarity with known proteins from neutrophilic organisms, including conservation of functional motifs, permitting their identification by bioinformatics tools and allowing the recognition of common themes in iron transport across distantly related species. However, significant differences in amino acid sequence were detected in pertinent domains that suggest ways in which the periplasmic and outer membrane proteins of A. ferrooxidans maintain structural integrity and relevant protein-protein contacts at low pH. Unexpectedly, the microorganism also contains candidate genes, organized in operon-like structures that potentially encode at least 11 siderophore systems for the uptake of Fe(III), although it does not exhibit genes that could encode the biosynthesis of the siderophores themselves. The presence of multiple Fe(III) uptake systems suggests that A. ferrooxidans can inhabit aerobic environments where iron is scarce and where siderophore producers are present. It may also help to explain why it cannot tolerate high Fe(III) concentrations in bioleaching operations where it is out-competed by Leptospirillum species.

     

Iron-dependent growth of and siderophore production by two heterotrophic bacteria isolated from brackish water of the southern Baltic Sea

Iron is indispensable to the growth and metabolism of all marine organisms, including bacteria. In this work, we investigated and compared the influence of iron(III) concentration on the growth of and siderophore production by two heterotrophic bacteria – Micrococcus luteus and Bacillus silvestris.Our results showed that the iron concentration strongly influences the growth of both species. The growth curves were different for each iron concentration and each strain. M. luteus grew more rapidly than B. silvestris, but produced a roughly four times smaller quantity of siderophores. Both M. luteus and B. silvestris secreted hydroxamate-type siderophores and α-keto/α-hydroxy acids, but did not produce catecholates.This paper is probably the first to report on siderophore production by B. silvestris and M. luteus isolated from seawater. Moreover, the influence of different iron concentrations on the growth of and siderophore production in these bacteria has been documented. This provides further evidence indicating iron bioavailability as the actual reason for siderophore release by biota.     

Hydroxamate Production as a High Affinity Iron Acquisition Mechanism in Paracoccidioides Spp

Iron is a micronutrient required by almost all living organisms, including fungi. Although this metal is abundant, its bioavailability is low either in aerobic environments or within mammalian hosts. As a consequence, pathogenic microorganisms evolved high affinity iron acquisition mechanisms which include the production and uptake of siderophores. Here we investigated the utilization of these molecules by species of the Paracoccidioides genus, the causative agents of a systemic mycosis. It was demonstrated that iron starvation induces the expression of Paracoccidioides ortholog genes for siderophore biosynthesis and transport. Reversed-phase HPLC analysis revealed that the fungus produces and secretes coprogen B, which generates dimerumic acid as a breakdown product. Ferricrocin and ferrichrome C were detected in Paracoccidioides as the intracellular produced siderophores. Moreover, the fungus is also able to grow in presence of siderophores as the only iron sources, demonstrating that beyond producing, Paracoccidioides is also able to utilize siderophores for growth, including the xenosiderophore ferrioxamine. Exposure to exogenous ferrioxamine and dimerumic acid increased fungus survival during co-cultivation with macrophages indicating that these molecules play a role during host-pathogen interaction. Furthermore, cross-feeding experiments revealed that Paracoccidioides siderophores promotes growth of Aspergillus nidulans strain unable to produce these iron chelators. Together, these data denote that synthesis and utilization of siderophores is a mechanism used by Paracoccidioides to surpass iron limitation. As iron paucity is found within the host, siderophore production may be related to fungus pathogenicity.     

Isolation and identification of ferrioxamine G and E inHafnia alvei

Summary Under conditions of iron-deprivationHafnia alvei (Enterobacteriaceae) produces ferrioxamine G as the principal siderophore. Maximum hydroxamate siderophore production occurred at medium iron limitation. The ferrioxamines were extracted, purified by gel filtration and chromatography on silica gel yielding a major and a minor siderophore fraction. The minor siderophore fraction contained three siderophores, among which ferrioxamine E could be identified by HPLC and FAB mass spectrometry. Reductive hydrolysis of the ferrioxamine G fraction yielded succinic acid and a mixture of diaminopentane and diaminobutane, as determined by gas-liquid chromatography and GLC/MS. HPLC and FAB mass spectrometry confirmed that the ferrioxamine G fraction consisted of two different species, G₁ and G₂, possessing molecular masses of 671 Da and 658 Da respectively.     

Isolation of bacteria producing siderophores under alkaline conditions

Summary The isolation of bacteria producing siderophores under alkaline conditions is reported. Enrichment cultures initiated with samples from a number of alkaline environmental sources yielded 80 isolates. From this group selections were made on the basis of growth at high pH and the gallium-binding capacity of the siderophores. It was found that some isolates grew well and high concentrations of siderophore were detected whereas others grew well in the presence of much lower concentrations of siderophore. The effect of iron, gallium and aluminium on growth and siderophore production in batch culture was investigated for six isolates. The presence of iron greatly decreased the siderophore concentration in these cultures, whereas the response to added gallium or aluminium was dependent upon the isolate. Offsprint requests to: D. J. Gascoyne     

The purple non‐sulfur bacterium Rhodopseudomonas palustris produces novel petrobactin‐related siderophores under aerobic and anaerobic conditions

Many bacteria produce siderophores to bind and take up Fe(III), an essential trace metal with extremely low solubility in oxygenated environments at circumneutral pH. The purple non‐sulfur bacterium Rhodopseudomonas palustris str. CGA009 is a metabolically versatile model organism with high iron requirements that is able to grow under aerobic and anaerobic conditions. Siderophore biosynthesis has been predicted by genomic analysis, however, siderophore structures were not identified. Here, we elucidate the structure of two novel siderophores from R. palustris: rhodopetrobactin A and B. Rhodopetrobactins are structural analogues of the known siderophore petrobactin in which the Fe chelating moieties are conserved, including two 3,4‐dihydroxybenzoate and a citrate substructure. In the place of two spermidine linker groups in petrobactin, rhodopetrobactins contain two 4,4′‐diaminodibutylamine groups of which one or both are acetylated at the central amine. We analyse siderophore production under different growth modes and show that rhodopetrobactins are produced in response to Fe limitation under aerobic as well as under anaerobic conditions. Evaluation of the chemical characteristics of rhodopetrobactins indicates that they are well suited to support Fe acquisition under variable oxygen and light conditions.     

Elucidation of the complete ferrichrome A biosynthetic pathway in Ustilago maydis

Iron is an important element for many essential processes in living organisms. To acquire iron, the basidiomycete Ustilago maydis synthesizes the iron‐chelating siderophores ferrichrome and ferrichrome A. The chemical structures of these siderophores have been elucidated long time ago but so far only two enzymes involved in their biosynthesis have been described. Sid1, an ornithine monoxygenase, is needed for the biosynthesis of both siderophores, and Sid2, a non‐ribosomal peptide synthetase (NRPS), is involved in ferrichrome generation. In this work we identified four novel enzymes, Fer3, Fer4, Fer5 and Hcs1, involved in ferrichrome A biosynthesis in U. maydis. By HPLC‐MS analysis of siderophore accumulation in culture supernatants of deletion strains, we show that Fer3, an NRPS, Fer4, an enoyl‐coenzyme A (CoA)‐hydratase, and Fer5, an acylase, are required for ferrichrome A production. We demonstrate by conditional expression of the hydroxymethyl glutaryl (HMG)‐CoA synthase Hcs1 in U. maydis that HMG‐CoA is an essential precursor for ferrichrome A. In addition, we heterologously expressed and purified Hcs1, Fer4 and Fer5, and demonstrated the enzymatic activities by in vitro experiments. Thus, we describe the first complete fungal siderophore biosynthetic pathway by functionally characterizing four novel genes responsible for ferrichrome A biosynthesis in U. maydis.     

Ferrichrome in <Emphasis Type="Italic">Schizosaccharomyces pombe</Emphasis> – an iron transport and iron storage compound

Schizosaccharomyces pombe has been assumed not to produce siderophores. Nevertheless, the genomic sequence of this fission yeast revealed the presence of siderophore biosynthetic genes for hydroxamates. Applying a bioassay based on an Aspergillus nidulans strain deficient in siderophore biosynthesis, and using reversed-phase HPLC and mass spectrometry analysis, we demonstrate that S. pombe excretes and accumulates intracellularly the hydroxamate-type siderophore ferrichrome. Under iron-limiting conditions, the cellular ferrichrome pool was present in the desferri-form, while under iron-rich conditions, in the ferri-form. In contrast to S. pombe, hydroxamate-type siderophores could not be detected in two other yeast species, Saccharomyces cerevisiae and Candida albicans.     

Multiple modes of iron uptake by the filamentous,siderophore‐producing cyanobacterium,Anabaena sp. PCC 7120

Iron is a member of a small group of nutrients that limits aquatic primary production. Mechanisms for utilizing iron have to be efficient and adapted according to the ecological niche. In respect to iron acquisition cyanobacteria, prokaryotic oxygen evolving photosynthetic organisms can be divided into siderophore‐ and non‐siderophore‐producing strains. The results presented in this paper suggest that the situation is far more complex. To understand the bioavailability of different iron substrates and the advantages of various uptake strategies, we examined iron uptake mechanisms in the siderophore‐producing cyanobacterium Anabaena sp. PCC 7120. Comparison of the uptake of iron complexed with exogenous (desferrioxamine B, DFB) or to self‐secreted (schizokinen) siderophores by Anabaena sp. revealed that uptake of the endogenous produced siderophore complexed to iron is more efficient. In addition, Anabaena sp. is able to take up dissolved, ferric iron hydroxide species (Fe′) via a reductive mechanism. Thus, Anabaena sp. exhibits both, siderophore‐ and non‐siderophore‐mediated iron uptake. While assimilation of Fe′ and FeDFB are not induced by iron starvation, FeSchizokinen uptake rates increase with increasing iron starvation. Consequently, we suggest that Fe′ reduction and uptake is advantageous for low‐density cultures, while at higher densities siderophore uptake is preferred.     

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.