Trishydroxamates and triscatecholates based on monosaccharides and myo-inositol as artificial siderophores

New trishydroxamates and triscatecholates based on methyl -D-glucopyranoside, methyl -D-galactopyranoside, methyl -D-ribopyranoside and methyl -D-xylopyranoside as well as on 1,3,5-tri-O-benzyl-myo-inositol were synthesized. N-Methylsuccinohydroxamate, N-methylglutarohydroxamate and their O-benzoyl derivatives were used as hydroxamate moieties. 2,3-Dihydroxybenzoyl derivatives and acylated compounds as well as 2,3- and 3,4-dihydroxybenzylidenehydrazino derivatives, partly with spacer groups, were utilized as catecholate components. The siderophore activity of the prepared siderophore analogues was examined by a growth promotion assay with various Gram-negative bacteria and mycobacteria and by the CAS-assay. Some trishydroxamates and triscatecholates showed siderophore activity on Gram-negative bacteria and triscatecholates on mycobacteria. Iron complexes of the trishydroxamates act as siderophores for all types of iron transport mutants. The recognition and uptake specificity of these compounds was studied by E. coli siderophore receptor and iron transport mutants. Structure activity correlations are discussed.     

Iron uptake in pseudorevertants of Escherichia coli K-12 mutants with multiple defects in the enterochelin system

Iron uptake in pseudorevertants of Escherichia coli K-12 strains which lack the ability to synthesize enterochelin, 2,3-dihydroxybenzoate, and the ferrienterochelin receptor protein was characterized. In four independent pseudorevertants, the suppressor mutations which permitted growth in iron-poor environments appeared to be located in ompB, the regulatory locus for the porin proteins. Unlike wild-type cells, the pseudorevertants were unable to utilize ferrienterochelin and could acquire iron from citrate without induction by prior growth in citrate. The energy requirements of the pseudorevertant system appeared to be identical to those of the enterochelin system. Evidence that loss of the porin proteins results in the secretion by the pseudorevertants of a molecule with siderophore activity is presented; this siderophore is able to remove iron from the non-biological iron chelators nitrilotriacetic acid and , -dipyridyl but not from the siderophores ferrichrome and enterochelin.     

Quorum-sensing antagonist (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone influences siderophore biosynthesis in Pseudomonas putida and Pseudomonas aeruginosa

Siderophore synthesis of Pseudomonas putida F1 was found to be regulated by quorum sensing since normalized siderophore production (per cell) increased 4.2-fold with cell density after the cells entered middle exponential phase; similarly, normalized siderophore concentrations in Pseudomonas aeruginosa JB2 increased 28-fold, and a 5.5-fold increase was seen for P. aeruginosa PAO1. Further evidence of the link between quorum sensing and siderophore synthesis of P. putida F1 was that the quorum-sensing-disrupter (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the marine red alga Delisea pulchra was found to inhibit the formation of the siderophore produced by P. putida F1 in a concentration-dependent manner, with 57% siderophore synthesis repressed by 100 g/ml furanone. In contrast, this furanone did not affect the siderophore synthesis of Burkholderia cepacia G4 at 20–40 g/ml, and stimulated siderophore synthesis of P. aeruginosa JB2 2.5- to 3.7-fold at 20–100 g/ml. Similarly, 100 g/ml furanone stimulated siderophore synthesis in P. aeruginosa PAO1 about 3.5-fold. The furanone appears to interact with the quorum-sensing machinery of P. aeruginosa PAO1 since it stimulates less siderophore synthesis in the P. aeruginosa qscR quorum-sensing mutant (QscR is a negative regulator of LasI, an acylated homoserine lactone synthase).     

Isolation and partial characterization of siderophore mutants ofAzotobacter vinelandii

Azotobacter vinelandii was mutagenized with ethyl methanesulfonate, and colonies that did not produce the fluorescent yellow-green pigment that is characteristic of the wild type were selected. All 32 stable nonfluorescent mutants failed to secrete the siderophore azotobactin and were also impaired to some extent in the production of the second majorA. vinelandii siderophore, azotochelin. Mutants also showed differences in their capacity to grow on medium supplemented with either 200 M bipyridyl or 200 M Fe (III). In the absence of iron, an 84-kilodalton outer membrane protein, which is a major derepressed component, was missing in some of the mutants. Thus, siderophore production inA. vinelandii appears to be a highly integrated system in which the syntheses of azotobactin and azotochelin are functionally coupled.     

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 siderophore produced by <Emphasis Type="Italic">Pseudomonas syringae</Emphasis> BAF.1 and its inhibitory effects on spore germination and mycelium morphology of <Emphasis Type="Italic">Fusarium oxysporum</Emphasis>

In this study, an antagonistic bacterium against Fusarium oxysporum was identified and designated as Pseudomonas syringae strain BAF.1 on the basis of 16S rDNA sequence analysis and physiological-biochemical characteristics. It produced catechol-species siderophore at a molecular weight of 488.59 Da and a maximum amount of 55.27 μg/ml with glucose as a carbon source and asparagine as a nitrogen source at a C/N ratio of 10:1, 30°C and pH 7. The siderophore exhibited prominent antagonistic activity against Fusarium oxysporum with a maximum inhibition rate of 95.24% and had also suppressive effects on other kinds of 11 phytopathogenic fungi in the absence of FeCl₃·6H₂O. Spore germination was completely inhibited by 50 μl of the siderophorecontaining solution, and the ultrastructures of mycelia and spores were also considerably suppressed by siderophore treatment as established by electron microscopy observation. These results indicate that the siderophore produced by Pseudomonas syringae BAF.1 could be potentially used for biocontrol of pathogenic Fusarium oxysporum.     

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

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

The nutritional selectivity of a siderophore-catabolizing bacterium

The ability of a siderophore-catabolizing bacterium to assimilate ferric ion was examined. While the bacterium utilizes the siderophore deferrioxamine B (DFB) as a carbon source, it was incapable of using the ferricion analogue (ferrioxamine B) as an iron source. It did, however, assimilate the ferric ion of the chelator ferric nitrilotriacetic acid and of the siderophore ferrirhodotorulic acid (ferriRA). Neither ferriRA nor its deferrated analog (RA), however, were capable of functioning as carbon sources for the bacterium. The microbe thus employs a nutritional selectivity with respect to these two siderophores. That is, it does not use the siderophore it employs as a carbon source (DFB) as an iron source nor does the siderophore utilized as an iron source, i.e. ferriRA, nor its deferrated analog (RA), serve as carbon sources for the organism.This paper is dedicated to the memory of Professor Thomas Emery. Professor Emery was instrumental in giving support and advice at a time when such mentorship greatly aided the corresponding author in developing a program concerning the catabolism of siderophores by microbes.     

Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily

Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 fet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with ⁵⁵Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.     

Hydroxamate siderophore synthesis by Phialocephala fortinii, a typical dark septate fungal root endophyte

The siderophore production of various isolates of Phialocephala fortinii was assessed quantitatively as well as qualitatively in batch assays under pure culture conditions at different pH values and iron(III) concentrations. We found a distinct effect of both of these parameters on siderophore synthesis and as well as on fungal growth. In comparative analyses of two of the isolates, maximum siderophore production was found at a pH in the range of pH 4.0 to 4.5 while, under the experimental conditions employed, the optimal concentration of ferric iron was determined to be between 20–40 g iron (III) l^–1 (0.36–0.72 M, respectively). HPLC analysis of the culture filtrate of most of the isolates of P. fortinii revealed the excretion of ferricrocin as main hydroxamate siderophore, followed by ferrirubin and ferrichrome C. The pattern of release of these three substances proved to be dependent on pH and iron(III) concentration of the culture medium, and to be specific for each isolate under investigation.     

A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae

While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3 background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using ⁵⁵Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.     

Isolation and identification of the principal siderophore of the plant pathogenic fungusBotrytis cinerea

Summary The plant pathogenic hyphomyceteBotrytis cinerea has been shown to produce several trihydroxamate siderophores under conditions of low-iron stress. The total siderophores amounted to approximately 30 mg/l culture filtrate after 5 days of incubation in an asparagine/salt/glucose medium. Thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) on a reversed phase indicated that ferrirhodin is the predominant siderophore of this fungus. Chemical characterization of the principal siderophore by fast-atom-bombardment (FAB) mass spectrometry, nuclear magnetic resonance (¹H-NMR,¹³C-NMR) and comparison with a reference revealed the identity with ferrirhodin. NMR studies performed on desferrirhodin (desferrirhodin) in dimethylsulfoxide and water revealed the existence of two conformers in D₂O resulting from acis-trans isomerization of the hydroxamic acid groups. Comparative iron-uptake studies showed the following order of uptake inB. cinerea: ferrichrysin (100%), ferrirubin (57%), ferrirhodin (45%), hexahydroferrirhodin (45%), coprogen 6%. Concentration-dependent uptake of ferrirhodin resulted in saturation kinetics only in the low concentration range of 0–30 M (K _m = 2.5 M,V _max = 80 pmol min^–1 mg(^–1). A non-saturable, linear uptake was observed in the high concentration range of 30–80 M. The low concentration range appears to be the physiologically significant range, where siderophore-mediated iron transport inB. cinerea occurs.     

N α -Dimethylcoprogens Three novel trihydroxamate siderophores from pathogenic fungi

Summary Three novel siderophores have been isolated from a highly pathogenic strain ofAlternaria longipes (ATCC 26293). The compounds are N -dimethylated analogs of coprogen, neocoprogen I and isoneocoprogen I. Structures of the compounds have been determined by¹H- and¹³C-NMR, fast-atom-bombardment (FAB) mass spectroscopy and partial hydrolysis. One of the new compounds, N -dimethylcoprogen, is also produced, as the major siderophore, in another fungus,Fusarium dimerum.     

The structure of partly decondensed metaphase chromosomes

The technique of freeze-drying was applied to examine the submicroscopic organisation of metaphase chromosomes from Chinese hamster after removal of bivalent cations with EDTA and removal of histone HI with 0,6 M NaCl. Treated chromosomes increased in size, and nucleosomal filaments appeared at the periphery of the chromosomes. Removal of bivalent cations is accompanied with the appearance of regularly organized structures of the beads-on-a-string type. The regular organization of the fibers is damaged as soon as histone H1 is removed. After decondensation in a 0,6 M NaCl solution the metaphase chromosomes were treated with staphylococcal nuclease in situ on EM grids and the residual structures analysed using electron microscopy. Nucleohistone fibers were visible at the periphery of the chromosomes at the beginning of digestion. After complete elimination of the nucleohistone fibers in the course of digestion the remaining proteinaceous material was represented by aggregates of irregular shape and of varying size. These were either concentrated along the central axis of the chromatids or, at the final step of digestion, scattered evenly over the entire area that had been occupied by the chromosome. Presumably, in the chromosome prior to digestion, the material did not form an integral protein structure similar to a scaffold in dehistonised and spread chromosomes. An alternative interpretation for the fragmentation of protein material in the chromosome considers possible degradation of the protein scaffold in the course of digestion.     

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.     

Aspergillus fumigatus SidJ Mediates Intracellular Siderophore Hydrolysis

Siderophore-mediated iron handling is crucial for the virulence of Aspergillus fumigatus. Here we identified a new component of its siderophore metabolism, termed SidJ, which is encoded by AFUA_3G03390. The encoding gene is localized in a siderophore biosynthetic gene cluster that is conserved in a variety of fungi. During iron starvation, SidJ deficiency resulted in decreased growth and increased intracellular accumulation of hydrolysis products of the siderophore fusarinine C. The implied role in siderophore hydrolysis is consistent with a putative esterase domain in SidJ, which now represents the first functionally characterized member of the DUF1749 (domain of unknown function) protein family, with members found exclusively in fungi and plants.     

Silver staining the chromosome scaffold

Cytological silver-staining procedures reveal the presence of a core running along the chromatid axes of isolated HeLa mitotic chromosomes. In this communication we examine the relationship between this core and the nonhistone chromosome scaffolding, isolated and characterized in previous publications from this laboratory. When chromosomes on coverslips were subjected to the steps used for scaffold isolation in vitro and subsequently stained with silver, the characteristic core staining was unaffected. Control experiments suggested that the core does not contain large amounts of DNA. When scaffolds were isolated in vitro, centrifuged onto electron microscope grids, and stained with silver, they were found to stain selectively under conditions where specific core staining was observed in intact chromosomes. These results suggest that the nonhistone scaffolding is the principal target of the silver stain in chromosomes.     

Siderophore production by Pseudomonas stutzeri under aerobic and anaerobic conditions

The siderophore production of the facultative anaerobe Pseudomonas stutzeri, strain CCUG 36651, grown under both aerobic and anaerobic conditions, was investigated by liquid chromatography and mass spectrometry. The bacterial strain has been isolated at a 626-m depth at the Äspö Hard Rock Laboratory, where experiments concerning the geological disposal of nuclear waste are performed. In bacterial culture extracts, the iron in the siderophore complexes was replaced by gallium to facilitate siderophore identification by mass spectrometry. P. stutzeri was shown to produce ferrioxamine E (nocardamine) as the main siderophore together with ferrioxamine G and two cyclic ferrioxamines having molecular masses 14 and 28 atomic mass units lower than that of ferrioxamine E, suggested to be ferrioxamine D₂ and ferrioxamine X₁, respectively. In contrast, no siderophores were observed from anaerobically grown P. stutzeri. None of the siderophores produced by aerobically grown P. stutzeri were found in anaerobic natural water samples from the Äspö Hard Rock Laboratory.In order to facilitate iron(III) acquisition, plants and microorganisms, such as fungi and bacteria, produce and excrete strong iron(III) chelators, i.e., siderophores (18, 22, 23, 33, 34). While fungal siderophores bind to iron(III) by hydroxamate ligands, bacterial siderophores are more structurally diverse, and common ligands are catecholates, hydroxamates, and carboxylates (21). The iron(III) stability constants for bacterial siderophores vary in the range of 10²⁰ to 10⁵² (6). In addition to iron(III), other metals can be complexed by siderophores. For the trihydroxamate siderophore desferrioxamine B, sometimes called proferrioxamine B (10), some actinides have been shown to have stability constants in the same range as the ferric stability constant (10^30.6), e.g., 10^26.6 with thorium(IV) and 10^30.8 with plutonium(VI) (32), while the stability constant for uranium(VI) was lower, i.e., 10¹⁸ (2).Concerning bacteria, there are several reports on siderophore production by Pseudomonas spp. (1, 3, 4, 19). More than 50 structurally related siderophores, i.e., pyoverdins, produced by the fluorescent Pseudomonas spp., especially Pseudomonas fluorescens and Pseudomonas aeruginosa, have been characterized (3). All pyoverdins emit yellow fluorescent light due to the presence of a 5-amino-2,3-dihydro-8,9-dihydroxy-1-H-pyrimido-quinoline-carboxylic chromophore, to which a peptide chain and a carboxyl chain are attached (1, 3). Nonfluorescent Pseudomonas has also been shown to produce siderophores, such as ferrioxamine E, also called nocardamine (Fig. ​(Fig.1),1), which was produced by one strain of Pseudomonas stutzeri (19). In addition to ferrioxamines, the P. stutzeri strain KC produced a smaller siderophore, i.e., pyridine-2,6-bis(thiocarboxylic acid) (35). Conversely, a catecholate-type siderophore was shown to be produced by another strain of P. stutzeri, which did not produce any hydroxamate siderophores (4).Open in a separate windowFIG. 1.Structures, molecular masses (mw), and stability constants (K_s) of ferric complexes of the three ferrioxamines: ferrioxamine B (B), ferrioxamine E (E), and ferrioxamine G (G) (5, 18).Most of the studies on bacterial siderophore production have been conducted on microorganisms growing under aerobic conditions. One field-based report, however, indicates the occurrence of putative siderophores in anaerobic environments also (29). In the present study, siderophore production has been studied with both aerobic and anaerobic cultures of P. stutzeri. This species is a facultative aerobe, able to grow with oxygen or nitrate as the electron acceptor, meaning that it can be active under both anaerobic and aerobic conditions. The P. stutzeri strain CCUG 36651, studied here, has been isolated from a depth of 626 m below ground at the Äspö Hard Rock Laboratory (16), where research concerning the geological disposal of nuclear waste is performed. The possibility of mobilizing radionuclides by complexing compounds from bacteria is an important research area in the context of nuclear waste disposal research. It is unknown if such compounds are produced in aquifers under conditions relevant to a disposal site, which would be approximately 500 m underground in granitic rock (27).A study from 2004 shows that P. stutzeri growing aerobically in the presence of uranium-containing shale leached Fe, Mo, V, and Cr from the shale material (17). More recently it was shown that the supernatant of aerobically and anaerobically cultured P. stutzeri was able to increase the partitioning of added Fe, Pm, Am, and Th into the aqueous phase in samples where quartz sand was used as a solid surface (16). Aerobic supernatants maintained 60% or more of the added metals in solution, while anaerobic supernatants were best at maintaining Am in solution, reaching a value of 40% in solution. The increased partitioning to the aqueous phase in the presence of the supernatants was ascribed to the production of organic ligands. Supernatants of both aerobically and anaerobically grown P. stutzeri strain CCUG 36651 yielded a positive response on the universal siderophore assay, the CAS assay (16). This assay is based on ligand competition for iron bound to the colored chrome azurol complex (25, 30).In this study, siderophore production by P. stutzeri strain CCUG 36651 was investigated using mass spectrometry (MS) and liquid chromatography (LC) followed by mass spectrometric detection. Electrospray ionization mass spectrometry (ESI-MS) and electrospray ionization tandem mass spectrometry (ESI-MS/MS) are useful tools in characterizing siderophores such as ferrioxamines (10, 13, 14, 28, 31). In order to detect iron(III)-chelating compounds, the ferric iron can be replaced by gallium(III) through ascorbate-mediated reduction of iron(III) (8, 20). In mass spectra, gallium-bound substances are easily recognized due to the characteristic isotope pattern of gallium, where the intensity of the ⁷¹Ga signal is about 66% of that of the ⁶⁹Ga signal. The use of ESI provides so-called soft ionization; thus, information about the molecular weight is obtained. However, by employing MS/MS, fragmentation is achieved, providing more information about the compound structure.In order to verify the chemical difference between the siderophores found by ESI-MS, chromatographic separation was performed. In this case, one reversed-phase C₁₈ column and one column containing a porous graphitic carbon (PGC) stationary phase were used. The separation mechanism of PGC is a combination of hydrophobic interactions, as in C₁₈, and electrostatic interactions between π-electrons. In order to detect substances at low concentrations, column-switched capillary chromatography with MS detection was used. The detection limits of the combined LC-MS/MS system used in this study are in the range of 1 to 5 nM for hydroxamate siderophores of the ferrichrome and ferrioxamine families (9). In order to facilitate analysis of lower concentrations of ferrioxamines, natural water samples were preconcentrated by solid-phase extraction (SPE), resulting in minimum detectable concentrations in the range of 0.02 to 0.1 nM, depending on the initial sample volume.     

Characterization of ferrioxamine E as the principal siderophore ofErwinia herbicola (Enterobacter agglomerans)

Summary Several strains of the enterobacterial groupErwinia herbicola (Enterobacter agglomerans) were screened for siderophore production. After 3 days of growth in a low-iron medium, all strains studied produced hydroxamate siderophores. The retention values of the main siderophore during thin-layer chromatography on silica gel plates and on HPLC reversed-phase columns were identical with those of an authentic sample of ferrioxamine E (norcardamine). Gas-chromatographic analysis of the HI hydrolyzate yielded succinic acid and 1,5-diaminopentane in equimolar amounts; fast-atom-bombardment (FAB) mass spectroscopy showed a molecular mass of 653 Da. Iron from⁵⁵Fe-labelled ferrioxamine E was well taken up by iron-starved cells ofE. herbicola (K _m=0.1 M,V _max=8 pmol mg^–1 min^–1). However, besides ferrioxamine E (100%), several exogenous siderophores such as enterobactin (94.5%), ferric citrate (78.5%), coprogen (63.5%) and ferrichrome (17.5%) served as siderophores, suggesting the presence of multiple siderophore receptors in the outer membrane ofE. herbicola.