Elucidation of crude siderophore extracts from supernatants of Pseudomonas sp. ZnCd2003 cultivated in nutrient broth supplemented with Zn, Cd, and Zn plus Cd.

This research aimed to study siderophores secreted from Pseudomonas sp. PDMZnCd2003, a Zn/Cd tolerant bacterium. The effects of Zn and/or Cd stress were examined in nutrient broth to achieve the actual environmental conditions. Acid and alkali supernatants and liquid-liquid extraction with ethyl acetate and butanol were carried out to obtain crude extracts containing different amounts of the metals. The bacterial growth, UV-visible spectra of the supernatants and siderophore production indicated that the production of siderophores tended to be linked to primary metabolites. Pyocyanin was produced in all treatments, while pyoverdine was induced by stress from the metals, especially Cd. FT-IR spectra showed C=O groups and sulfur functional groups that were involved in binding with the metals. LC-MS revealed that pyocyanin, 1-hydroxy phenazine, pyoverdine, and pyochelin were present in the crude extracts. S K-edge XANES spectra showed that the main sulfur species in the extracts were the reduced forms of sulfide, thiol, and disulfide, and their oxidation states were affected by coordination with Zn and/or Cd. In addition, Zn K-edge EXAFS spectra and Cd K-edge EXAFS spectra presented Zn-O and Cd-O as coordination in the first shell, in case the extracts contained less metal. Although the mix O/S ligands had chelation bonding with Zn and Cd in the other extracts. For the role of S groups in pyochelin binding with the metals, this was the first report. The results of these experiments could be extended to Pseudomonas that respond to metal contaminated environments.

Lipopeptide production by Bacillus atrophaeus strain B44 and its biocontrol efficacy against cotton rhizoctoniosis.

OBJECTIVES: An assay was conducted to show the comparisons the effects of nine metal ions on antagonistic metabolites (lipopeptides, siderophores and gibberellins) by Bacillus atrophaeus strain B44 using well-diffusion assays, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis, chrome azurol S plus mannitol salt agar (CAS-MSA) tests, and reversed-phase high-performance liquid chromatography (RP-HPLC) analysis. This assay is also designed to demonstrate the biocontrol efficacy of B44 against cotton rhizoctoniosis using pot culture tests. RESULTS: Both the lipopeptide yield and the antimicrobial activity of B44 increase with the MnSO4, MgSO4, CaCO3, and CuSO4 treatments and either have no effect or decreased lipopeptide yield and antimicrobial activity with the FeSO4, K2HPO4, KCl, KH2PO4 and ZnSO4 treatments. The medium containing MgSO4 has no significant effect on either the lipopeptide yield or antimicrobial activity. MALDI-TOF-MS analysis shows a broad range of m/z peaks, indicating that strain B44 produces a complex mixture of iturin, surfactin, and fengycin lipopeptides. Gibberellin production by strain B44 varies greatly depending on the culture medium, and the siderophore production is not significantly affected by the culture medium. Pot tests show that lipopeptide production affects the disease control efficacy of strain B44. CONCLUSION: The biocontrol efficacy of B. atrophaeus strain B44 is related to the lipopeptide yield. Moreover, B. atrophaeus strain B44 significantly increases the size of cotton seedlings, which is related to the GA3 concentration.

Rhizobactin B is the preferred siderophore by a novel Pseudomonas isolate to obtain iron from dissolved organic matter in peatlands.

Bacteria often release diverse iron-chelating compounds called siderophores to scavenge iron from the environment for many essential biological processes. In peatlands, where the biogeochemical cycle of iron and dissolved organic matter (DOM) are coupled, bacterial iron acquisition can be challenging even at high total iron concentrations. We found that the bacterium Pseudomonas sp. FEN, isolated from an Fe-rich peatland in the Northern Bavarian Fichtelgebirge (Germany), released an unprecedented siderophore for its genus. High-resolution mass spectrometry (HR-MS) using metal isotope-coded profiling (MICP), MS/MS experiments, and nuclear magnetic resonance spectroscopy (NMR) identified the amino polycarboxylic acid rhizobactin and a novel derivative at even higher amounts, which was named rhizobactin B. Interestingly, pyoverdine-like siderophores, typical for this genus, were not detected. With peat water extract (PWE), studies revealed that rhizobactin B could acquire Fe complexed by DOM, potentially through a TonB-dependent transporter, implying a higher Fe binding constant of rhizobactin B than DOM. The further uptake of Fe-rhizobactin B by Pseudomonas sp. FEN suggested its role as a siderophore. Rhizobactin B can complex several other metals, including Al, Cu, Mo, and Zn. The study demonstrates that the utilization of rhizobactin B can increase the Fe availability for Pseudomonas sp. FEN through ligand exchange with Fe-DOM, which has implications for the biogeochemical cycling of Fe in this peatland.

Novel South African Rare Actinomycete Kribbella speibonae Strain SK5: A Prolific Producer of Hydroxamate Siderophores Including New Dehydroxylated Congeners.

In this paper, we report on the chemistry of the rare South African Actinomycete Kribbella speibonae strain SK5, a prolific producer of hydroxamate siderophores and their congeners. Two new analogues, dehydroxylated desferrioxamines, speibonoxamine 1 and desoxy-desferrioxamine D1 2, have been isolated, together with four known hydroxamates, desferrioxamine D1 3, desferrioxamine B 4, desoxy-nocardamine 5 and nocardamine 6, and a diketopiperazine (DKP) 7. The structures of 1-7 were characterized by the analysis of HRESIMS and 1D and 2D NMR data, as well as by comparison with the relevant literature. Three new dehydroxy desferrioxamine derivatives 8-10 were tentatively identified in the molecular network of K. speibonae strain SK5 extracts, and structures were proposed based on their MS/MS fragmentation patterns. A plausible spb biosynthetic pathway was proposed. To the best of our knowledge, this is the first report of the isolation of desferrioxamines from the actinobacterial genus Kribbella.

Siderophores in plant root tissue: Tagetes patula nana colonized by the arbuscular mycorrhizal fungus Gigaspora margarita.

More than 70% of vascular plant species live in symbiosis with arbuscular mycorrhizal (AM) fungi. In addition to other effects this symbiosis is known for its significance for plant nutrition including iron. Fungal iron mobilization from soil is commonly dependent on siderophores. This study reports on a search for such iron-chelators in root tissue of Tagetes patula nana var. plena colonized by Gigaspora margarita. The AM colonized plants and uninoculated controls were grown under strictly axenic conditions. HPLC analyses of aqueous extracts from plant roots have provided clear evidence for the presence of a rhizoferrin type siderophore, named glomuferrin, in root tissue of mycorrhizal seedlings. Results from HPLC analytical work are seconded by molecular biological data: A BLASTp search revealed that the AM fungal species Gigaspora rosea, Rhizophagus irregularis (formerly Glomus intraradices), Glomus cerebriformis and Diversispora epigea encode a non-ribosomal peptide synthetase (NRPS)-independent siderophore synthase (NIS), which is homologous to the rhizoferrin synthetase of Rhizopus delemar. Thus this study indicates that the biosynthesis of rhizoferrin type siderophores such as glomuferrin (= bis-imidorhizoferrin) may be widespread in the AM symbiosis.

Characterization of the promiscuous N-acyl CoA transferase, LgoC, in legonoxamine biosynthesis.

More than 500 siderophores are known to date, but only three were identified to be aryl-containing hydroxamate siderophores, legonoxamines A and B from Streptomyces sp. MA37, and aryl ferrioxamine 2 from Micrococcus luteus KLE1011. Siderophores are produced by microorganisms to scavenge iron from the environment, thereby making this essential metal nutrient available to the microbe. We demonstrate here that LgoC from MA37 is responsible for the key aryl-hydroxamate forming step in legonoxamine biosynthesis. Biochemical characterization established that LgoC displays considerable promiscuity for the acylation between N-hydroxy-cadaverine and SNAC (N-acetylcysteamines) thioester derivatives.

Siderophores from the Entomopathogenic Fungus Beauveria bassiana.

We report NMR- and MS-based structural characterizations of siderophores and related compounds from Beauveria bassiana (Balsamo-Crivelli) Vuillemin, including ten new chemical entities (2-4, 6-9, 11-12, and 15) and five known compounds, (1, 5, 10, 13, and 14). The siderophore mixture from ARSEF strain #2680 included two compounds in which N5-mevalonyl-N5-hydroxyornithine replaces both (2) or one (3) of the N5-anhydromevalonyl-N5-hydroxyornithine units of dimerumic acid (1). Mevalonolactone (14) was present as a degradation product of 2 and 3. ARSEF #2860 also produced compounds that have mannopyranose (5, 6) or 4-O-methyl-mannopyranose units (4, 7), two compounds (8, 9) that can be rationalized as 4-O-methyl-mannopyranosyl analogues of the esterifying acid moieties of metachelins A and B, respectively, and two probable decomposition products of 1, a nitro compound (11) and a formate (12). Beauverichelin A (15), a coprogen-type siderophore that represents the di-4-O-methyl-mannopyranosyl analogue of metachelin A, was detected in crude extracts of ARSEF #2860, but only in trace amounts. ARSEF strains #252 and #1955 yielded beauverichelin A in quantities that were sufficient for NMR analysis. Only the di- (1-7) and trihydroxamate (15) siderophores showed iron-binding activity in the CAS assay and, when ferrated, showed strong ESIMS signals consistent with 1:1 ligand/iron complexes.

Chemistry and Biology of Siderophores from Marine Microbes.

Microbial siderophores are multidentate Fe(III) chelators used by microbes during siderophore-mediated assimilation. They possess high affinity and selectivity for Fe(III). Among them, marine siderophore-mediated microbial iron uptake allows marine microbes to proliferate and survive in the iron-deficient marine environments. Due to their unique iron(III)-chelating properties, delivery system, structural diversity, and therapeutic potential, marine microbial siderophores have great potential for further development of various drug conjugates for antibiotic-resistant bacteria therapy or as a target for inhibiting siderophore virulence factors to develop novel broad-spectrum antibiotics. This review covers siderophores derived from marine microbes.

Nocardamin glucuronide, a new member of the ferrioxamine siderophores isolated from the ascamycin-producing strain Streptomyces sp. 80H647.

A new siderophore glucuronide, nocardamin glucuronide (1), was isolated together with nocardamin (2) by applying the one strain-many compounds (OSMAC) approach to the ascamycin-producing strain, Streptomyces sp. 80H647, and performing multivariate analysis using mass spectral data. Structure elucidation was accomplished by a combination of NMR and MS analyses. The absolute configuration of the glucuronic acid moiety was found to be ß-D-GlcA by hydrolysis using ß-glucuronidase, subsequent derivatization of the hydrolysate, and comparison with standards. The siderophore activity of 1 was evaluated through the chrome azurol S assay and was comparable to that of 2 and deferoxamine (IC50 13.4, 9.5, and 6.3 µM, respectively). Nocardamin glucuronide (1) is the first example of a siderophore glucuronide.

Madurastatin D1 and D2, Oxazoline Containing Siderophores Isolated from an Actinomadura sp.

Two new siderophores, madurastatin D1 and D2, together with (-)-madurastatin C1, the enantiomer of a known compound, were isolated from marine-derived Actinomadura sp. The presence of an unusual 4-imidazolidinone ring in madurastatins D1 and D2 inspired us to sequence the Actinomadura sp. genome and to identify the mad biosynthetic gene cluster, knowledge of which enables us to now propose a biosynthetic pathway. Madurastatin D1 and D2 are moderately active in antimicrobial assays with M. luteus.

Characterization of Aspergillus niger siderophore that mediates bioleaching of rare earth elements from phosphorites.

Siderophores are extra-cellular inducible compounds produced by aerobic microorganisms and plants to overcome iron insolubility via its chelation and then uptake inside the cell. This work aims to study the characteristics of siderophore that is produced by a rhizosphere-inhabiting fungus. This fungus has been morphologically and molecularly identified as Aspergillus niger with the ability to produce 87% siderophore units. The obtained siderophore in PDB medium gave a positive result with tetrazolium test and a characteristic spectrum with a maximum absorbance at 450 nm in FeCl3 test that did not shift in response to different pH degrees (5-9). This indicates that the obtained siderophore is a trihydroxymate in nature. After purification, the FTIR and NMR analyses showed that the obtained siderophore is considered to be ferrichrome. The purified siderophore has been further evaluated as a tool to extract uranium, thorium and rare earth elements (REEs) from Egyptian phosphorites obtained from Abu Tartur Mine area. The inductively coupled plasma atomic emission spectroscopy analysis showed that the highest removal efficiency percentage was for uranium (69.5%), followed by samarium (66.7%), thorium (55%), lanthanum (51%), and cerium (50.1%). This result confirmed the ability of hydroxymate siderophores to chelate the aforementioned precious elements, a result that paves the way for bioleaching to replace abiotic techniques in order to save the cost of such elements in an environmentally friendly way.

A suite of asymmetric citrate siderophores isolated from a marine Shewanella species.

Woodybactins A-D are a suite of new fatty acyl siderophores produced by the luminous marine bacterium Shewanella woodyi MS32. While this bacterium has a set of genes homologous to the biosynthetic gene cluster for aerobactin, aerobactin is not produced. The arrangement of these genes within the genome differs in S. woodyi MS32 when compared to E. coli and other species producing siderophores similar to aerobactin, and one synthetase gene which would append a second acyl-hydroxylysine to the terminal carboxylate of citrate is not functional. Within the suite of woodybactins A-D, which differ by the fatty acid appendage, one contains an unusual C9 (9:0) fatty acid and one contains a unique branched C9 iso (9:0 iso) fatty acid, as well as a C8 (8:0) and C10 (10:0) fatty acid.

Analysis of Microbial Siderophores by Mass Spectrometry.

Siderophores represent important microbial virulence factors and infection biomarkers. Their monitoring in fermentation broths, bodily fluids, and tissues should be reproducible. Similar isolation, characterization, and quantitation studies can often have conflicting results, and without proper documentation of sample collection, data processing, and analysis methods, it is difficult to reexamine the data and reconcile these differences. In this Springer Nature Protocol, we present the procedure optimized for ferricrocin/triacetylfusarinine C extraction from biological material as well as for tissue fixation and cryosectioning for optical microscopy and for both elemental and molecular mass spectrometry imaging. Special attention is paid to siderophore data mining from conventional and product ion mass spectra, liquid chromatography, and mass spectrometry imaging datasets, performed here by our free software called CycloBranch.

Labrenzbactin from a coral-associated bacterium Labrenzia sp.

A new catecholate-containing siderophore, labrenzbactin (1), was isolated from the fermentation broth of a coral-associated bacterium Labrenzia sp. The structure and absolute configuration of 1 was determined by spectroscopic methods and Marfey's analysis. Overall, 1 showed antimicrobial activity against Ralstonia solanacearum SUPP1541 and Micrococcus luteus ATCC9341 with MIC values of 25 and 50 µg ml-1, respectively, and cytotoxicity against P388 murine leukemia cells with an IC50 of 13 µM.

Metabolomics Assay Identified a Novel Virulence-Associated Siderophore Encoded by the High-Pathogenicity Island in Uropathogenic Escherichia coli.

To date, yersiniabactin remains the only identified siderophore encoded by the high pathogenicity island (HPI) in uropathogenic Escherichia coli (UPEC). In the present study, we aim to discover and identify new siderophores in the HPI-dependent biosynthetic pathway using a combinational strategy of metabolomics and genetics. A global metabolome assay of wild-type UTI89, UTI89ΔybtS, and UTI89ΔybtS with the substrate addition of salicylic acid found numerous unknown metabolite features that were encoded by the HPI with an obvious substrate dependency on salicylic acid. One metabolite feature with m/ z 307.0206 was shown to have a similar phenotype as yersiniabactin. Furthermore, isotope mass spectrum calculations and MS/MS annotations were combined to identify this metabolite as HPTzTn-COOH. HPTzTn-COOH was verified as a new siderophore in this study, and it was observed to have a robust capacity to chelate different metals, including Al3+, Ni2+, and Ca2+, in addition to binding Fe3+. Our data revealed that HPTzTn-COOH has a stronger diagnostic ability over the more conventionally used yersiniabactin, as characterized by its high production throughout UPEC strains harboring HPI. Altogether, our discoveries revise the siderophore family, and HPTzTn-COOH can be classified as an additional key siderophore along with yersiniabactin.

Isolation and Characterization of the Acinetobactin and Baumannoferrin Siderophores Produced by Acinetobacter baumannii.

Siderophores are high-affinity iron chelators produced and used by bacteria to prosper under iron-limiting conditions they normally encounter in the environment and hosts. In this chapter, we describe the isolation and purification of the siderophores acinetobactin and baumannoferrin produced by the bacterial pathogen Acinetobacter baumannii using XAD-7 batch adsorption and high-pressure liquid chromatography (HPLC). We also describe chemical tests and biological assays used to detect the presence of catechol and hydroxamate siderophores in culture supernatants, XAD-7 extracts, and HPLC fractions.

Analysis of Iron Requirements and Siderophore Production.

This chapter describes the methods for inducing, detecting, and purifying the Legionella pneumophila siderophore. The first protocol details the methods by which L. pneumophila is cultured to facilitate production of the siderophore, rhizoferrin. This chapter then describes how to purify siderophore from culture supernatants through sequential reversed-phase/weak-anion exchange chromatography and high-performance liquid chromatography. The next section describes assays which allow the detection of the iron-binding capability and the biological activity of the purified siderophore. Lastly, this chapter describes the growth of L. pneumophila in chemically defined liquid medium (CDM) containing various iron sources as a method to assess the iron requirements of L. pneumophila.

Proteomic Response to Thaxtomin Phytotoxin Elicitor Cellobiose and to Deletion of Cellulose Utilization Regulator CebR in Streptomyces scabies.

Streptomyces scabies is responsible for common scab disease on root and tuber vegetables. Production of its main phytotoxin thaxtomin A is triggered upon transport of cellulose byproducts cellotriose and cellobiose, which disable the repression of the thaxtomin biosynthesis activator gene txtR by the cellulose utilization regulator CebR. To assess the intracellular response under conditions where S. scabies develops a virulent behavior, we performed a comparative proteomic analysis of wild-type S. scabies 87-22 and its cebR null mutant (hyper-virulent phenotype) grown in the absence or presence of cellobiose. Our study revealed significant changes in abundance of proteins belonging to metabolic pathways known or predicted to be involved in pathogenicity of S. scabies. Among these, we identified proteins of the cello-oligosaccharide-mediated induction of thaxtomin production, the starch utilization system required for utilization of the carbohydrate stored in S. scabies's hosts, and siderophore synthesis utilization systems, which are key features of pathogens to acquire iron once they colonized the host. Thus, proteomic analysis supported by targeted mass spectrometry-based metabolite quantitative analysis revealed the central role of CebR as a regulator of virulence of S. scabies.

Chemical characterization and ligand behaviour of Pseudomonas veronii 2E siderophores.

Siderophores are low-molecular weight ligands secreted by bacteria as a survival strategy in Fe(III)-lacking environments. They bind not only Fe(III), but Co(II), Zn(II), Mn(II), Ni(II), Ga(III) as a detoxification alternative. The synthesis, purification and characterization of siderophores produced by Pseudomonas veronii 2E were evaluated to be applied in future environmental technologies. Optimal production was obtained in Fe(III)-free M9-succinate at 25 °C, 40 h and pH 6.9. Siderophores were chemically characterized as hydroxamate and catechol mixed-type. Spectroscopic analysis indicated their belonging to the pyoverdine family, behaving as ligand to Cd(II), Zn(II), Cu(II), Ni(II) and Cr(III), which promoted siderophoregenesis during growth. Siderophore-Cd(II) complexation was studied by electrochemical monitored titration revealing one family of moderate-strength binding sites. Mass spectral analysis evidenced the secretion of a variety of molecules (molecular mass ca.1200 u). Non pathogenic Pseudomonas veronii 2E siderophores represent a safe alternative for the concrete application of environmental technologies and clinical procedures.

Gramibactin is a bacterial siderophore with a diazeniumdiolate ligand system.

Genome mining and chemical analyses revealed that rhizosphere bacteria (Paraburkholderia graminis) produce a new type of siderophore, gramibactin, a lipodepsipeptide that efficiently binds iron with a logß value of 27.6. Complexation-induced proton NMR chemical shifts show that the unusual N-nitrosohydroxylamine (diazeniumdiolate) moieties participate in metal binding. Gramibactin biosynthesis genes are conserved in numerous plant-associated bacteria associated with rice, wheat, and maize, which may utilize iron from the complex.