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BACKGROUND: Prior to soil formation, phosphate liberated by rock weathering is often sequestered into highly insoluble lanthanide phosphate minerals. Dissolution of these minerals releases phosphate and lanthanides to the biosphere. Currently, the microorganisms involved in phosphate mineral dissolution and the role of lanthanides in microbial metabolism are poorly understood. RESULTS: Although there have been many studies of soil microbiology, very little research has investigated microbiomes of weathered rock. Here, we sampled weathered granite and associated soil to identify the zones of lanthanide phosphate mineral solubilisation and genomically define the organisms implicated in lanthanide utilisation. We reconstructed 136 genomes from 11 bacterial phyla and found that gene clusters implicated in lanthanide-based metabolism of methanol (primarily xoxF3 and xoxF5) are surprisingly common in microbial communities in moderately weathered granite. Notably, xoxF3 systems were found in Verrucomicrobia for the first time, and in Acidobacteria, Gemmatimonadetes and Alphaproteobacteria. The xoxF-containing gene clusters are shared by diverse Acidobacteria and Gemmatimonadetes, and include conserved hypothetical proteins and transporters not associated with the few well studied xoxF systems. Given that siderophore-like molecules that strongly bind lanthanides may be required to solubilise lanthanide phosphates, it is notable that candidate metallophore biosynthesis systems were most prevalent in bacteria in moderately weathered rock, especially in Acidobacteria with lanthanide-based systems. CONCLUSIONS: Phosphate mineral dissolution, putative metallophore production and lanthanide utilisation by enzymes involved in methanol oxidation linked to carbonic acid production co-occur in the zone of moderate granite weathering. In combination, these microbial processes likely accelerate the conversion of granitic rock to soil.

Ribosomally synthesized and posttranslationally modified peptides (RiPPs) are a structurally diverse group of natural products that bacteria employ in their survival strategies. Herein, we characterized the structure, the biosynthetic pathway, and the mode of action of a RiPP family called bufferins. With thousands of homologous biosynthetic gene clusters throughout the bacterial phylogenetic tree, bufferins form by far the largest family of RiPPs modified by multinuclear nonheme iron-dependent oxidases (MNIO, DUF692 family). Using Caulobacter vibrioides bufferins as a model, we showed that the conserved Cys residues of their precursors are transformed into 5-thiooxazoles, further expanding the reaction range of MNIO enzymes. This rare modification is installed in conjunction with a partner protein of the DUF2063 family. Bufferin precursors are rare examples of bacterial RiPPs found to feature an N-terminal Sec signal peptide allowing them to be exported by the ubiquitous Sec pathway. We reveal that bufferins are involved in copper homeostasis, and their metal-binding propensity requires the thiooxazole heterocycles. Bufferins enhance bacterial growth under copper stress by complexing excess metal ions. Our study thus describes a large family of RiPP metallophores and unveils a widespread but overlooked metal homeostasis mechanism in bacteria. [ABSTRACT FROM AUTHOR]

ABSTRACT Limiting the availability of transition metals at infection sites serves as a critical defense mechanism employed by the innate immune system to combat microbial infections. Pseudomonas aeruginosa exhibits a remarkable ability to thrive in zinc-deficient environments, facilitated by intricate cellular responses governed by numerous genes regulated by the zinc-responsive transcription factor Zur. Many of these genes have unknown functions, including those within the predicted PA2911-PA2914 and PA4063-PA4066 operons. A structural bioinformatics investigation revealed that PA2911-PA2914 comprises a TonB-dependent outer membrane receptor and inner membrane ABC-permeases responsible for importing metal-chelating molecules, whereas PA4063-PA4066 contains genes encoding a MacB transporter, likely involved in the export of large molecules. Molecular genetics and biochemical experiments, feeding assays, and intracellular metal content measurements support the hypothesis that PA2911-PA2914 and PA4063-PA4066 are engaged in the import and export of the pyochelin-cobalt complex, respectively. Notably, cobalt can reduce zinc demand and promote the growth of P. aeruginosa strains unable to import zinc, highlighting pyochelin-mediated cobalt import as a novel bacterial strategy to counteract zinc deficiency. These results unveil an unexpected role for pyochelin in zinc homeostasis and challenge the traditional view of this metallophore exclusively as an iron transporter.IMPORTANCEThe mechanisms underlying the remarkable ability of Pseudomonas aeruginosa to resist the zinc sequestration mechanisms implemented by the vertebrate innate immune system to control bacterial infections are still far from being fully understood. This study reveals that the Zur-regulated gene clusters PA2911-2914 and PA4063-PA4066 encode systems for the import and export of cobalt-bound pyochelin, respectively. This proves to be a useful strategy to counteract conditions of severe zinc deficiency since cobalt can replace zinc in many proteins. The discovery that pyochelin may contribute to cellular responses to zinc deficiency leads to a reevaluation of the paradigm that pyochelin is a siderophore involved exclusively in iron acquisition and suggests that this molecule has a broader role in modulating the homeostasis of multiple metals.

Abstract Background Prior to soil formation, phosphate liberated by rock weathering is often sequestered into highly insoluble lanthanide phosphate minerals. Dissolution of these minerals releases phosphate and lanthanides to the biosphere. Currently, the microorganisms involved in phosphate mineral dissolution and the role of lanthanides in microbial metabolism are poorly understood. Results Although there have been many studies of soil microbiology, very little research has investigated microbiomes of weathered rock. Here, we sampled weathered granite and associated soil to identify the zones of lanthanide phosphate mineral solubilisation and genomically define the organisms implicated in lanthanide utilisation. We reconstructed 136 genomes from 11 bacterial phyla and found that gene clusters implicated in lanthanide-based metabolism of methanol (primarily xoxF3 and xoxF5) are surprisingly common in microbial communities in moderately weathered granite. Notably, xoxF3 systems were found in Verrucomicrobia for the first time, and in Acidobacteria, Gemmatimonadetes and Alphaproteobacteria. The xoxF-containing gene clusters are shared by diverse Acidobacteria and Gemmatimonadetes, and include conserved hypothetical proteins and transporters not associated with the few well studied xoxF systems. Given that siderophore-like molecules that strongly bind lanthanides may be required to solubilise lanthanide phosphates, it is notable that candidate metallophore biosynthesis systems were most prevalent in bacteria in moderately weathered rock, especially in Acidobacteria with lanthanide-based systems. Conclusions Phosphate mineral dissolution, putative metallophore production and lanthanide utilisation by enzymes involved in methanol oxidation linked to carbonic acid production co-occur in the zone of moderate granite weathering. In combination, these microbial processes likely accelerate the conversion of granitic rock to soil.

Siderophores are iron chelators and low-molecular-weight compounds secreted by various microorganisms under low-iron conditions. Many microorganisms produce siderophores in the natural environment as iron is an essential element for many of them. CAS assays are widely used to detect siderophores in cultures of various microorganisms; however, it is necessary to improve their sensitivity for the efficient application to fastidious microorganisms. We developed a simple, high-throughput CAS assay employing a buffer-free CAS reagent and diluted growth medium (10% dR2A) in a 96-well microplate. Using a diluted growth medium in agar plates suitable for iron-restricted conditions supported siderophore production by microorganisms from activated sludge. A buffer-free CAS reagent combined with a diluted growth medium revealed that these microorganisms tended to produce more siderophores or iron chelators than microorganisms under iron-rich conditions. Moreover, this buffer-free CAS assay easily and efficiently detected not only siderophore production but also the growth of fastidious microorganisms. [ABSTRACT FROM AUTHOR]

The field of microbe–metal interactions has been gaining significant attention. While the direct impact of metal oxyanions on bacteria has been investigated, significantly less attention has been placed on the ability of certain microbes to 'collect' such metal ions via secreted proteins. Many bacteria possess low-weight molecules called siderophores, which collect Fe from the environment to be brought back to the cell. However, some appear to have additional roles, including binding other metals, termed 'metallophores'. Microbes can remove/sequester these from their surroundings, but the breadth of those that can be removed is still unknown. Using the Chromeazurol S assay, we identified eight isolates, most belonging to the genus Pseudomonas, possessing siderophore activity, mainly from sites impacted by coal mine drainage, also possessing a metallophore activity toward the rare earth elements that does not appear to be related to ionic radii or previously reported EC50 concentrations for E. coli. We found the strength of metallophore activity towards these elements was as follows: Pr > Sc > Eu > Tm > Tb > Er > Yb > Ce > Lu > Sm > Ho > La > Nd > Dy > Gd > Y. This is the first study to investigate such activity and indicates bacteria may provide a means of removal/recovery of these critical elements. [ABSTRACT FROM AUTHOR]

Infection metallomics is a mass spectrometry (MS) platform we established based on the central concept that microbial metallophores are specific, sensitive, noninvasive, and promising biomarkers of invasive infectious diseases. Here we review the in vitro, in vivo, and clinical applications of metallophores from historical and functional perspectives, and identify under‐studied and emerging application areas with high diagnostic potential for the post‐COVID era. MS with isotope data filtering is fundamental to infection metallomics; it has been used to study the interplay between "frenemies" in hosts and to monitor the dynamic response of the microbiome to antibiotic and antimycotic therapies. During infection in critically ill patients, the hostile environment of the host's body activates secondary bacterial, mycobacterial, and fungal metabolism, leading to the production of metallophores that increase the pathogen's chance of survival in the host. MS can reveal the structures, stability, and threshold concentrations of these metal‐containing microbial biomarkers of infection in humans and model organisms, and can discriminate invasive disease from benign colonization based on well‐defined thresholds distinguishing proliferation from the colonization steady state. [ABSTRACT FROM AUTHOR]

Aspergillus fumigatus has been designated by the World Health Organization as a critical priority fungal pathogen. Some commercially available diagnostics for many forms of aspergillosis rely on fungal metabolites. These encompass intracellular molecules, cell wall components, and extracellular secretomes. This review summarizes the shortcomings of antibody tests compared to tests of fungal products in body fluids and highlights the application of β-d-glucan, galactomannan, and pentraxin 3 in bronchoalveolar lavage fluids. We also discuss the detection of nucleic acids and next-generation sequencing, along with newer studies on Aspergillus metallophores. [ABSTRACT FROM AUTHOR]

To survive in the metal-scarce environment within the host, pathogens synthesize various small molecular metallophores to facilitate the acquisition of transition metals. The cobalt and nickel transporter (Cnt) system synthesizes and transports staphylopine, a nicotianamine-like metallophore, and serves as a primary transition metal uptake system in Gram-positive bacteria including the human pathogen Staphylococcus aureus. In this study, we report the design of the first inhibitor of the Cnt system by targeting the key aminobutanoyltransferase CntL which is involved in the biosynthesis of staphylopine. Through structure-guided fragment linking and optimization, a class of acceptor-adenosine dual-site inhibitors against S. aureus CntL (Sa CntL) were designed and synthesized. The most potent inhibitor, compound 9 , demonstrated a Δ T m value of 9.4 °C, a K d value of 0.021 ± 0.004 μM, and an IC 50 value of 0.06 μM against Sa CntL. The detailed mechanism by which compound 9 inhibits Sa CntL has been elucidated through a high-resolution co-crystal structure. Treatment with compound 9 resulted in a moderate downregulation of intracellular concentrations of iron, nickel, and cobalt ions in the S. aureus cells cultured in the metal-scarce medium, providing the first chemical validation of the important role of Cnt system in bacterial metal acquisition. Our findings pave the way for the development of CntL-based antibacterial agents in future. [Display omitted] • Structure-guided fragment linking led to the first inhibitors of the Cnt system. • The most potent compound 9 demonstrated nanomolar activity against S. aureus CntL. • Dual-site inhibitory mechanism of 9 to CntL was elucidated by cocrystal structure. • Treatment with 9 downregulated transition metal levels in S. aureus cells. [ABSTRACT FROM AUTHOR]

Clinical isolates of Yersinia, Klebsiella, and Escherichia coli frequently secrete the small molecule metallophore yersiniabactin (Ybt), which passivates and scavenges transition metals during human infections. YbtT is encoded within the Ybt biosynthetic operon and is critical for full Ybt production in bacteria. However, its biosynthetic function has been unclear because it is not essential for Ybt production by the in vitro reconstituted nonribosomal peptide synthetase/polyketide synthase (NRPS/PKS) pathway. Here, we report the structural and biochemical characterization of YbtT. YbtT structures at 1.4-1.9 Å resolution possess a serine hydrolase catalytic triad and an associated substrate chamber with features similar to those previously reported for low-specificity type II thioesterases (TEIIs). We found that YbtT interacts with the two major Ybt biosynthetic proteins, HMWP1 (high-molecular-weight protein 1) and HMWP2 (high-molecular-weight protein 2), and hydrolyzes a variety of aromatic and acyl groups from their phosphopantetheinylated carrier protein domains. In vivo YbtT titration in uropathogenic E. coli revealed a distinct optimum for Ybt production consistent with a tradeoff between clearing both stalled inhibitory intermediates and productive Ybt precursors from HMWP1 and HMWP2. These results are consistent with a model in which YbtT maintains cellular Ybt biosynthesis by removing nonproductive, inhibitory thioesters that form aberrantly at multiple sites on HMWP1 and HMWP2.

Inflammatory bowel disease (IBD)-associated fibrosis causes significant morbidity. Mechanisms are poorly understood but implicate the microbiota, especially adherent-invasive Escherichia coli (AIEC). We previously demonstrated that AIEC producing the metallophore yersiniabactin (Ybt) promotes intestinal fibrosis in an IBD mouse model. Since macrophages interpret microbial signals and influence inflammation/tissue remodeling, we hypothesized that Ybt metal sequestration disrupts this process. Here, we show that macrophages are abundant in human IBD-fibrosis tissue and mouse fibrotic lesions, where they co-localize with AIEC. Ybt induces profibrotic gene expression in macrophages via stabilization and nuclear translocation of hypoxia-inducible factor 1-alpha (HIF-1α), a metal-dependent immune regulator. Importantly, Ybt-producing AIEC deplete macrophage intracellular zinc and stabilize HIF-1α through inhibition of zinc-dependent HIF-1α hydroxylation. HIF-1α+ macrophages localize to sites of disease activity in human IBD-fibrosis strictures and mouse fibrotic lesions, highlighting their physiological relevance. Our findings reveal microbiota-mediated metal sequestration as a profibrotic trigger targeting macrophages in the inflamed intestine., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The field of microbe–metal interactions has been gaining significant attention. While the direct impact of metal oxyanions on bacteria has been investigated, significantly less attention has been placed on the ability of certain microbes to ‘collect’ such metal ions via secreted proteins. Many bacteria possess low-weight molecules called siderophores, which collect Fe from the environment to be brought back to the cell. However, some appear to have additional roles, including binding other metals, termed ‘metallophores’. Microbes can remove/sequester these from their surroundings, but the breadth of those that can be removed is still unknown. Using the Chromeazurol S assay, we identified eight isolates, most belonging to the genus Pseudomonas, possessing siderophore activity, mainly from sites impacted by coal mine drainage, also possessing a metallophore activity toward the rare earth elements that does not appear to be related to ionic radii or previously reported EC50 concentrations for E. coli. We found the strength of metallophore activity towards these elements was as follows: Pr > Sc > Eu > Tm > Tb > Er > Yb > Ce > Lu > Sm > Ho > La > Nd > Dy > Gd > Y. This is the first study to investigate such activity and indicates bacteria may provide a means of removal/recovery of these critical elements.

Mycobacterium tuberculosis (Mtb) endures a combination of metal scarcity and toxicity throughout the human infection cycle, contributing to complex clinical manifestations. Pathogens counteract this paradoxical dysmetallostasis by producing specialized metal trafficking systems. Capture of extracellular metal by siderophores is a widely accepted mode of iron acquisition, and Mtb iron-chelating siderophores, mycobactin, have been known since 1965. Currently, it is not known whether Mtb produces zinc scavenging molecules. Here, we characterize low-molecular-weight zinc-binding com- pounds secreted and imported by Mtb for zinc acquisition. These molecules, termed kupyaphores, are produced by a 10.8 kbp bio- synthetic cluster and consists of a dipeptide core of ornithine and phenylalaninol, where amino groups are acylated with isonitrile- containing fatty acyl chains. Kupyaphores are stringently regulated and support Mtb survival under both nutritional deprivation and intoxication conditions. A kupyaphore-deficient Mtb strain is unable to mobilize sufficient zinc and shows reduced fitness upon infection. We observed early induction of kupyaphores in Mtb-infected mice lungs after infection, and these metabolites dis- appeared after 2 wk. Furthermore, we identify an Mtb-encoded isonitrile hydratase, which can possibly mediate intracellular zinc release through covalent modification of the isonitrile group of kupyaphores. Mtb clinical strains also produce kupyaphores during early passages. Our study thus uncovers a previously unknown zinc acquisition strategy of Mtb that could modulate host–pathogen interactions and disease outcome. [ABSTRACT FROM AUTHOR]

Limiting the availability of transition metals at infection sites serves as a critical defense mechanism employed by the innate immune system to combat microbial infections. Pseudomonas aeruginosa exhibits a remarkable ability to thrive in zinc-deficient environments, facilitated by intricate cellular responses governed by numerous genes regulated by the zinc-responsive transcription factor Zur. Many of these genes have unknown functions, including those within the predicted PA2911-PA2914 and PA4063-PA4066 operons. A structural bioinformatics investigation revealed that PA2911-PA2914 comprises a TonB-dependent outer membrane receptor and inner membrane ABC-permeases responsible for importing metal-chelating molecules, whereas PA4063-PA4066 contains genes encoding a MacB transporter, likely involved in the export of large molecules. Molecular genetics and biochemical experiments, feeding assays, and intracellular metal content measurements support the hypothesis that PA2911-PA2914 and PA4063-PA4066 are engaged in the import and export of the pyochelin-cobalt complex, respectively. Notably, cobalt can reduce zinc demand and promote the growth of P. aeruginosa strains unable to import zinc, highlighting pyochelin-mediated cobalt import as a novel bacterial strategy to counteract zinc deficiency. These results unveil an unexpected role for pyochelin in zinc homeostasis and challenge the traditional view of this metallophore exclusively as an iron transporter., Importance: The mechanisms underlying the remarkable ability of Pseudomonas aeruginosa to resist the zinc sequestration mechanisms implemented by the vertebrate innate immune system to control bacterial infections are still far from being fully understood. This study reveals that the Zur-regulated gene clusters PA2911-2914 and PA4063-PA4066 encode systems for the import and export of cobalt-bound pyochelin, respectively. This proves to be a useful strategy to counteract conditions of severe zinc deficiency since cobalt can replace zinc in many proteins. The discovery that pyochelin may contribute to cellular responses to zinc deficiency leads to a reevaluation of the paradigm that pyochelin is a siderophore involved exclusively in iron acquisition and suggests that this molecule has a broader role in modulating the homeostasis of multiple metals., Competing Interests: The authors declare no conflict of interest.

New infectious diseases and increase in drug-resistant microbial pathogens emphasize the need for antibiotics with novel mode-of-action. Tetramates represented by fungi-derived tenuazonic acid and bacterial polycyclic tetramate macrolactams (PTMs) are an important family of natural products with a broad spectrum of antimicrobial activities. Despite their potential application as new antibiotics, it remains unknown how PTMs function. In this study, genomic mining revealed that PTM biosynthetic gene clusters (BGCs) are widespread in both Gram-positive and Gram-negative bacteria, and we investigated a sponge endosymbiont Actinoalloteichus hymeniacidonis harboring a potential PTM-BGC. Xanthobaccin A that previously has only been isolated from a Gram-negative bacterium was obtained after a scale-up fermentation, isolation, and structure elucidation through mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Xanthobaccin A as well as two previously reported tetramates, equisetin and ikarugamycin, exhibited antibacterial activities against Bacillus subtilis. In addition, these three tetramates were for the first time to be confirmed as metallophores and the stoichiometry of the complexes were shown to be Fe(III)(equisetin)3/Fe(III)(equisetin)2 and Fe(III)(ikarugamycin)2, respectively. Meanwhile, we found that all three tetramates could reduce ferric into ferrous iron, which triggers the Fenton chemistry reaction. Their antibacterial activity was reduced by adding the radical scavenger, vitamin C. Altogether, our work demonstrates that equisetin and PTMs can act as metallophores and their antimicrobial mechanism is possibly mediated through Fenton chemistry.

Multinuclear non-heme iron dependent oxidative enzymes (MNIOs), formerly known as domain of unknown function 692 (DUF692), are involved in the post-translational modification of peptides during the biosynthesis of peptide-based natural products. These enzymes catalyze highly unusual and diverse chemical modifications. Several class-defining features of this large family (>14 000 members) are beginning to emerge. Structurally, the enzymes are characterized by a TIM-barrel fold and a set of conserved residues for a di- or tri–iron binding site. They use molecular oxygen to modify peptide substrates, often in a four-electron oxidation taking place at a cysteine residue. This review summarizes the current understanding of MNIOs. Four modifications are discussed in detail: oxazolone-thioamide formation, β-carbon excision, hydantoin-macrocycle formation, and 5-thiooxazole formation. Briefly discussed are two other reactions that do not take place on Cys residues. [Display omitted] • Remarkable chemistry catalyzed by multinuclear non-heme iron-dependent oxidative enzymes (MNIOs). • MNIOs convert Cys to an oxazolone-thioamide in methanobactin biosynthesis and to 5-thiooxazole in bufferin biosynthesis. • MNIOs excise the β-carbon of Cys in 3-thiaglutamate biosynthesis. • MNIOs convert Cys residues into macrocyclic thioaminals and hydantoins. • MNIOs cleave the peptide backbone at Asn to give a C-terminal amide and convert a C-terminal Asp into aminopyruvic acid. [ABSTRACT FROM AUTHOR]

ABSTRACT Zinc is an essential nutrient in biological systems due to its structural or catalytic requirement in proteins involved in diverse cellular processes. To meet this cellular demand, microbes must acquire sufficient zinc from their environment. However, many environments have low zinc availability. One of the mechanisms used by bacteria to acquire zinc is through the production of small molecules known as zincophores. Similar to bacterial siderophores used for iron uptake, zincophores are synthesized by the bacterium and exported and then reimported as zincophore-zinc complexes. Thus far, only four zincophores have been described, including two from the human pathogens Staphylococcus aureus and Pseudomonas aeruginosa, in which they play a critical role in zinc acquisition during infection, and one in a soil bacterium. To determine what other microbes may produce zincophores, we used bioinformatic analyses to identify new zincophore biosynthetic gene clusters (BGCs) and predict the diversity of molecules synthesized. Genome neighborhood network analysis identified approximately 250 unique zincophore-producing species from actinobacteria, firmicutes, proteobacteria, and fusobacteria. This indicates that zincophores are produced by diverse bacteria that inhabit a broad range of ecological niches. Many of the BGCs likely produce characterized zincophores, based on similarity to the characterized systems. However, this analysis also identified numerous BGCs that, based on the colocalization of additional modifying enzymes and sequence divergence of the biosynthetic enzymes, are likely to produce unique zincophores. Collectively, these findings provide a comprehensive understanding of the zincophore biosynthetic landscape that will be invaluable for future research on these important small molecules. IMPORTANCE Bacteria must acquire essential nutrients, including zinc, from their environment. For bacterial pathogens, this necessitates overcoming the host metal-withholding response known as nutritional immunity. A novel type of zinc uptake mechanism that involves the bacterial production of a small zinc-scavenging molecule was recently described in the human pathogens Staphylococcus aureus, Pseudomonas aeruginosa, and Yersinia pestis, as well as the soil-associated bacterium Paenibacillus mucilaginosus. This suggests that zincophores may be important for zinc acquisition in diverse environments. In this study, we sought to identify other zincophore-producing bacteria using bioinformatics. We identified almost 250 unique zincophore-producing species, including human and animal pathogens, as well as isolates from soil, rhizosphere, plant, and marine habitats. Crucially, we observed diversity at the amino acid and gene organization levels, suggesting that many of these species are producing unique zincophores. Together, our findings highlight the importance of zincophores for a broad array of bacteria living in diverse environments.

Recent studies have shown that the metal adaptation of Actinobacteria offers a rich source of metal inducible environmentally relevant bio-compounds and molecules. These interact through biosorption towards the unique cell walls or via metal chelating activity of metallophors with trace elements, heavy metals and even with lanthanides to overcome limitations and toxic concentrations. Herein, the purpose is to investigate the adaptation potential of Gordonia rubripertincta CWB2 in dependence of the rare earths and to determine if we can utilize promising metallophore metal affinities for metal separation from aquatic solutions. For details on data interpretation and applicability of siderophores we refer to the related article entitled “Cultivation dependent formation of siderophores by Gordonia rubripertincta CWB2” [1].The respective workflow comprises a metal adaptation method to demonstrate effects on bacterial growth, pH, metallophore production, and metabolic change. All this was evaluated by LC-MS/MS and effects on biosorption of rare earths was verified by ICP-MS. Furthermore, we were able to carry out batch metal adsorption and desorption studies of metallophores entrapped in inorganic polymers of tetramethoxysilane (TMOS) to determine metal chelating capacities and selective enrichment effects from model solutions. The adaptation potential of strain CWB2 at increased erbium and manganese concentrations was verified by increased chelating activity on agar plates, in liquid assays and demonstrated by the successful enrichment of erbium by metallophore-functionalized TMOS-polymers from an aquatic model solution. Furthermore, the number of detected compounds in dependency of rare earths differ in spectral counts and diversity compared to the wild type. Finally, the biosorption of rare earths for the selected adaptation was increased significantly up to 2-fold compared to the wild-type. Overall a holistic approach to metal stress was utilised, integrating a bacterial erbium adaptation, metal chelating, biosorption of lanthanides and immobilization as well as enrichment of metals using metallophore functionalized inorganic TMOS polymers for separation of metals from aquatic model solutions.

To overcome the metal restriction imposed by the host's nutritional immunity, pathogenic bacteria use high metal affinity molecules called metallophores. Metallophore‐mediated metal uptake pathways necessitate complex cycles of synthesis, secretion, and recovery of the metallophore across the bacterial envelope. We recently discovered staphylopine and pseudopaline, two members of a new family of broad‐spectrum metallophores important for bacterial survival during infections. Here, we are expending the molecular understanding of the pseudopaline transport cycle across the diderm envelope of the Gram‐negative bacterium Pseudomonas aeruginosa. We first explored pseudopaline secretion by performing in vivo quantifications in various genetic backgrounds and revealed the specific involvement of the MexAB–OprM efflux pump in pseudopaline transport across the outer membrane. We then addressed the recovery part of the cycle by investigating the fate of the recaptured metal‐loaded pseudopaline. To do so, we combined in vitro reconstitution experiments and in vivo phenotyping in absence of pseudopaline transporters to reveal the existence of a pseudopaline modification mechanism, possibly involved in the metal release following pseudopaline recovery. Overall, our data allowed us to provide an improved molecular model of secretion, recovery, and fate of this important metallophore by P. aeruginosa. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Aerobic anoxygenic phototrophs have been isolated from a rich variety of environments including marine ecosystems, freshwater and meromictic lakes, hypersaline springs, and biological soil crusts, all in the hopes of understanding their ecological niche. Over 100 isolates were chosen for this study, representing 44 species from 27 genera. Interactions with Fe3+ and other metal(loid) cations such as Mg2+, V3+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Se4+ and Te2+ were tested using a chromeazurol S assay to detect siderophore or metallophore production, respectively. Representatives from 20 species in 14 genera of α-Proteobacteria, or 30% of strains, produced highly diffusible siderophores that could bind one or more metal(loid)s, with activity strength as follows: Fe > Zn > V > Te > Cu > Mn > Mg > Se > Ni > Co. In addition, γ-proteobacterial Chromocurvus halotolerans, strain EG19 excreted a brown compound into growth medium, which was purified and confirmed to act as a siderophore. It had an approximate size of ~341 Da and drew similarities to the siderophore rhodotorulic acid, a member of the hydroxamate group, previously found only among yeasts. This study is the first to discover siderophore production to be widespread among the aerobic anoxygenic phototrophs, which may be another key method of metal(loid) chelation and potential detoxification within their environments.

To guarantee the supply of critical elements in the future, the development of new technologies is essential. Siderophores have high potential in the recovery and recycling of valuable metals due to their metal-chelating properties. Using the Chrome azurol S assay, 75 bacterial strains were screened to obtain a high-yield siderophore with the ability to complex valuable critical metal ions. The siderophore production of the four selected strains Nocardioides simplex 3E, Pseudomonas chlororaphis DSM 50083, Variovorax paradoxus EPS, and Rhodococcus erythropolis B7g was optimized, resulting in significantly increased siderophore production of N. simplex and R. erythropolis. Produced siderophore amounts and velocities were highly dependent on the carbon source. The genomes of N. simplex and P. chlororaphis were sequenced. Bioinformatical analyses revealed the occurrence of an achromobactin and a pyoverdine gene cluster in P. chlororaphis, a heterobactin and a requichelin gene cluster in R. erythropolis, and a desferrioxamine gene cluster in N. simplex. Finally, the results of the previous metal-binding screening were validated by a proof-of-concept development for the recovery of metal ions from aqueous solutions utilizing C18 columns functionalized with siderophores. We demonstrated the recovery of the critical metal ions V(III), Ga(III), and In(III) from mixed metal solutions with immobilized siderophores of N. simplex and R. erythropolis.

Abstract The accessibility of iron (Fe) species for microbial processes is dependent on solubility and redox state, which are influenced by complexation with dissolved organic matter (DOM) and water-extractable organic matter (WEOM). We evaluated the complexation of these pools of organic matter to soluble Fe(II) and Fe(III) in the slightly acidic Schlöppnerbrunnen fen and subsequent effects on Fe(II) oxidation and Fe(III) reduction. We found the majority of soluble Fe(II) and Fe(III) is complexed to DOM. High-resolution mass spectrometry identified potential complexing partners in peat-derived water extracts (PWE), including compound classes known to function as ligands or electron shuttles, like tannins and sulfur-containing compounds. Furthermore, we observed clear differences in the stability of Fe(II)- and Fe(III)-DOM, with more labile complexes dominating the upper, oxic layers (0–10 cm) and more stable complexes in lower, anoxic layers (15–30 cm). Metal isotope-coded profiling identified a single potential chemical formula (C 42 H 57 O 13 N 9 Fe 2) associated with a stable Fe-DOM complex. Fe(III) reduction and Fe(II) oxidation incubations with Geobacter sulfurreducens PCA and Shewanella oneidensis MR-1 or Sideroxydans CL-21, respectively, were used to determine the influence of Fe-DOM complexes on Fe cycling rates. The addition of PWE led to a 2.3-fold increase in Fe(III) reduction rates and 0.5-fold increase in Fe(II) oxidation rates, indicating Fe-DOM complexes greatly influence microbial Fe cycling by potentially serving as electron shuttles. Molecular analyses revealed Fe(III)-reducing and Fe(II)-oxidizing bacteria co-exist across all depths, in approximately equal proportions (representing 0.1–1.0% of the total microbial community), despite observed changes in redox potential. The activity of Fe(III)-reducing bacteria might explain the presence of the detected Fe(II) stabilized via complexation with DOM even under oxic conditions in upper peat layers. Therefore, these Fe(II)-DOM complexes can be recycled by microaerophilic Fe(II)-oxidizers. Taken together, these results suggest Fe-DOM complexation in the fen accelerates microbial-mediated redox processes across the entire redox continuum. Graphical abstract Unlabelled Image Highlights • This field study of an Fe-rich peatland underlies the importance of Fe species determination. • The majority of Fe is complexed by DOM and keeps Fe(II) and Fe(III) in solution. • DOM stabilizes Fe(II) in oxic soil layers and contributes to the redox equilibria. • Fe-DOM-complexes enhance microbially-mediated Fe cycling. • Fe(II) and Fe(III) across all depths allows co-existence of Fe-cycling microorganisms. [ABSTRACT FROM AUTHOR]

Abstract The Actinobacterium Gordonia rubripertincta CWB2 (DSM 46758) produces hydroxamate-type siderophores (188 mg L−1) under iron limitation. Analytical reversed-phase HPLC allowed determining a single peak of ferric iron chelating compounds from culture broth which was confirmed by the Fe-CAS assay. Elution profile and its absorbance spectrum were similar to those of commercial (des)ferrioxamine B which was used as reference compound. This confirms previously made assumptions and shows for the first time that the genus Gordonia produces desferrioxamine-like siderophores. The reversed-phase HPLC protocol was optimized to separate metal-free and -loaded oxamines. This allowed to determine siderophore concentrations in solutions as well as metal affinity. The metal loading of oxamines was confirmed by ICP-MS. As a result, it was demonstrated that desferrioxamine prefers trivalent metal ions (Fe3+ > Ga3+ > V3+ > Al3+) over divalent ones. In addition, we aimed to show the applicability of the newly established reversed-phase HPLC protocol and to increase the re-usability of desferrioxamines as metal chelators by immobilization on mesocellular silica foam carriers. The siderophores obtained from strain CWB2 and commercial desferrioxamine B were successfully linked to the carrier with a high yield (up to 95%) which was verified by the HPLC method. Metal binding studies demonstrated that metals can be bound to non-immobilized and to the covalently linked desferrioxamines, but also to the carrier material itself. The latter was found to be unspecific and, therefore, the effect of the carrier material remains a field of future research. By means of a reversed CAS assay for various elements (Nd, Gd, La, Er, Al, Ga, V, Au, Fe, As) it was possible to demonstrate improved Ga3+- and Nd3+-binding to desferrioxamine loaded mesoporous silica carriers. The combination of the robust reversed-phase HPLC method and various CAS assays provides new avenues to screen for siderophore producing strains, and to control purification and immobilization of siderophores. [ABSTRACT FROM AUTHOR]

Copper-binding metallophores, or chalkophores, play a role in microbial copper homeostasis that is analogous to that of siderophores in iron homeostasis. The best-studied chalkophores are members of the methanobactin (Mbn) family-ribosomally produced, posttranslationally modified natural products first identified as copper chelators responsible for copper uptake in methane-oxidizing bacteria. To date, Mbns have been characterized exclusively in those species, but there is genomic evidence for their production in a much wider range of bacteria. This review addresses the current state of knowledge regarding the function, biosynthesis, transport, and regulation of Mbns. While the roles of several proteins in these processes are supported by substantial genetic and biochemical evidence, key aspects of Mbn manufacture, handling, and regulation remain unclear. In addition, other natural products that have been proposed to mediate copper uptake as well as metallophores that have biologically relevant roles involving copper binding, but not copper uptake, are discussed. [ABSTRACT FROM AUTHOR]

Natural products harbor unique and complex structures that provide valuable antibiotic scaffolds. With an increase in antibiotic resistance, natural products once again hold promise for new antimicrobial therapies, especially those with unique scaffolds that have been overlooked due to a lack of understanding of how they function. Dithiolopyrrolones (DTPs) are an underexplored class of disulfide-containing natural products, which exhibit potent antimicrobial activities against multidrug-resistant pathogens. DTPs were thought to target RNA polymerase, but conflicting observations leave the mechanisms elusive. Using a chemical genomics screen in Escherichia coli, we uncover a mode of action for DTPs-- the disruption of metal homeostasis. We show that holomycin, a prototypical DTP, is reductively activated, and reduced holomycin chelates zinc with high affinity. Examination of reduced holomycin against zinc-dependent metalloenzymes revealed that it inhibits E. coli class II fructose bisphosphate aldolase, but not RNA polymerase. Reduced holomycin also strongly inhibits metallo-β-lactamases in vitro, major contributors to clinical carbapenem resistance, by removing active site zinc. These results indicate that holomycin is an intracellular metal-chelating antibiotic that inhibits a subset of metalloenzymes and that RNA polymerase is unlikely to be the primary target. Our work establishes a link between the chemical structures of DTPs and their antimicrobial action; the ene-dithiol group of DTPs enables high-affinity metal binding as a central mechanism to inhibit metabolic processes. Our study also validates the use of chemical genomics in characterizing modes of actions of antibiotics and emphasizes the potential of metal-chelating natural products in antimicrobial therapy. [ABSTRACT FROM AUTHOR]

Siderophores, iron-scavenging small molecules, are fundamental to bacterial nutrient metal acquisition and enable pathogens to overcome challenges imposed by nutritional immunity. Multimodal imaging mass spectrometry allows visualization of host-pathogen iron competition, by mapping siderophores within infected tissue. We have observed heterogeneous distributions of Staphylococcus aureus siderophores across infectious foci, challenging the paradigm that the vertebrate host is a uniformly iron-depleted environment to invading microbes. [ABSTRACT FROM AUTHOR]

Trace metal limitation not only affects the biological function of organisms, but also the health of ecosystems and the global cycling of elements. The enzymatic machinery of microbes helps to drive critical biogeochemical cycles at the macroscale, and in many cases, the function of metalloenzyme-mediated processes may be limited by the scarcity of essential trace metals. In response to these nutrient limitations, some organisms employ a strategy of exuding metallophores, biogenic ligands that facilitate the uptake of metal ions. For example, bacterial, fungal, and graminaceous plant species are known to use Fe(III)-binding siderophores for nutrient acquisition, providing the best known and most thoroughly studied example of metallophores. However, recent breakthroughs have suggested or established the role of metallophores in the uptake of several other metallic nutrients. Furthermore, these metallophores may influence environmental trace metal fate and transport beyond nutrient acquisition. These discoveries have resulted in a deeper understanding of trace metal geochemistry and its relationship to the cycling of carbon and nitrogen in natural systems. In this review, we provide an overview of the current state of knowledge on the biogeochemistry of metallophores in trace metal acquisition, and explore established and potential metallophore systems. [ABSTRACT FROM AUTHOR]

Microbes are evolved to live in almost every environment and often produce small molecules to protect themselves, communicate, and sequester nutrients and minerals from their surroundings. Our recent discovery that the gold-associated microbe, Delftia acidovorans, overcomes Au toxicity through complexation and precipitation of soluble gold by the nonribosomal peptide metallophore, delftibactin, revealed a new mechanism for gold biomineralization and a possible industrial application for gold sequestration. In this work, we have sought to define more the scope of nanoparticle formation by other microbial metallophores and the physical properties of gold nanoparticles created by microbial natural products (hydroxamates, catechols, citrates, mixed ligands) and synthetics. [ABSTRACT FROM AUTHOR]

Nickel is an essential micronutrient for the survival of many microbes. On account of the toxicity of nickel and its scarcity in the environment, microbes have evolved specific systems for uptaking and delivering nickel to enzymes. NikA, the solute binding protein for the ATP-binding cassette (ABC) importer NikABCDE, plays a vital role in the nickel homeostasis of Escherichia coli by selectively binding nickel over other metals in the metabolically complex periplasm. While the endogenous ligand for NikA is known to be the Ni(II)-(L-His)2 complex, the molecular basis by which NikA selectively binds Ni(II)-(L-His)2 is unclear, especially considering that NikA can bind multiple metal-based ligands with comparable affinity. Here we show that, regardless of its promiscuous binding activity, NikA preferentially interacts with Ni(II)-(L-His)2, even over other metal-amino acid ligands with an identical coordination geometry for the metal. Replacing both the Ni(II) and the L-His residues in Ni(II)-(L-His)2 compromises binding of the ligand to NikA, in part because these alterations affect the degree by which NikA closes around the ligand. Replacing H416, the only NikA residue that ligates the Ni(II), with other potential metal-coordinating amino acids decreases the binding affinity of NikA for Ni(II)-(L-His)2 and compromises uptake of Ni(II) into E. coli cells, likely due to altered metal selectivity of the NikA mutants. Together, the biochemical and in vivo studies presented here define key aspects of how NikA selects for Ni(II)-(L-His)2 over other metal complexes, and can be used as a reference for studies into the metal selectivity of other microbial solute binding proteins., (© The Author(s) 2022. Published by Oxford University Press.)

Organic matter regulates the availability of important trace elements in aquatic and terrestrial ecosystems by acting as a source and container for microbes. To overcome the limitation of trace elements, nitrogen-fixing bacteria, e.g. release low-molecular-weight chelators (metallophores), which scavenge the essential cofactors of the nitrogenase, iron, and molybdenum (Mo), via complexation and subsequent uptake. The formation of metallophores is triggered by limiting conditions, which must be replicated in the laboratory in order to study metallophores as a mediator in metal cycling. While ethylenediaminetetraacetic acid (EDTA)-based buffer systems for metal cations are well established, there is limited knowledge regarding the buffering of oxoanions such as molybdate in a bacterial growth medium. To mimic the availability of molybdenum in nature under laboratory conditions, this study created a Mo-buffer system for bacterial growth media of the model organisms Azotobacter vinelandii and Frankia sp. CH37. We investigated selected hydroxypyridinones (HPs) as potential molybdenum-chelating agents, determining the amount required for efficient molybdenum complexation by calculating speciation plots of the various candidate complexes in artificial growth media at various pH values. The Mo-maltol system was identified as an ideal, nontoxic molybdenum-buffer system. In the presence of the Mo-maltol system, the growth of Frankia sp. was limited under diazotrophic conditions, whereas A. vinelandii could acquire molybdenum through the release of protochelin and subsequent molybdenum uptake. The study paves the way for unravelling molybdenum recruitment and homeostasis under limiting conditions in bacteria., (© The Author(s) 2022. Published by Oxford University Press.)

Two amide group containing pyridine derivatives, N -(pyridin-2-ylmethyl)picolinamide (PMPA) and N-(pyridin-2-ylmethyl)-2-((pyridin-2-ylmethyl)amino)acetamide (DPMGA), have been investigated as potential metallo-phores in the therapy of Alzheimer's disease. Their complex formation with Cu(II) and Zn(II) were characterized in details. Unexpectedly not only the Cu(II) but also the Zn(II) was able to induce deprotonation of the amide-NH, however, it occurred only at higher pH or at higher metal ion concentrations than the biological conditions. At μM concentration level mono complexes (MLH − 1) dominate with both ligands. Direct fluorescence and reactive oxygen species (ROS) producing measurements prove that both ligands are able to remove Cu(II) from its amyloid-β complexes (CuAβ). Correlation was also established between the conditional stability constant of the Cu(II) complexes with different ligands and their ability of inhibition of ROS production by CuAβ. A tridentate and a tetradentate neutral ligand containing amid group are able to prevent the reactive oxygen species production of Cu(II) complexes of amyloid-β. The Cu(II) complex of the tridentate one has optimal thermodynamic stability to become a metallophore, to reestablish copper homeostasis in Alzheimer's brain. Both ligands have high Cu(II)/Zn(II) specificity. [Display omitted] • Low molecular mass metallophores reestablish metal ion homeostasis in Alzheimer's brain. • Cu(II) and Zn(II) binding characterization of two amide containing pyridine derivatives. • Metallophores competing for Cu(II) with amyloid-β (Aβ) in the brain. • Correlation between β CuL and inhibition of reactive oxygen species production by Cu-Aβ. [ABSTRACT FROM AUTHOR]

New infectious diseases and increase in drug-resistant microbial pathogens emphasize the need for antibiotics with novel mode-of-action. Tetramates represented by fungi-derived tenuazonic acid and bacterial polycyclic tetramate macrolactams (PTMs) are an important family of natural products with a broad spectrum of antimicrobial activities. Despite their potential application as new antibiotics, it remains unknown how PTMs function. In this study, genomic mining revealed that PTM biosynthetic gene clusters (BGCs) are widespread in both Gram-positive and Gram-negative bacteria, and we investigated a sponge endosymbiont Actinoalloteichus hymeniacidonis harboring a potential PTM-BGC. Xanthobaccin A that previously has only been isolated from a Gram-negative bacterium was obtained after a scale-up fermentation, isolation, and structure elucidation through mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. Xanthobaccin A as well as two previously reported tetramates, equisetin and ikarugamycin, exhibited antibacterial activities against Bacillus subtilis . In addition, these three tetramates were for the first time to be confirmed as metallophores and the stoichiometry of the complexes were shown to be Fe(III)(equisetin) 3 /Fe(III)(equisetin) 2 and Fe(III)(ikarugamycin) 2 , respectively. Meanwhile, we found that all three tetramates could reduce ferric into ferrous iron, which triggers the Fenton chemistry reaction. Their antibacterial activity was reduced by adding the radical scavenger, vitamin C. Altogether, our work demonstrates that equisetin and PTMs can act as metallophores and their antimicrobial mechanism is possibly mediated through Fenton chemistry., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Ding, Zhang, Haidar, Bajimaya, Guo, Larsen and Gram.)

Staphylopine (StP) and other nicotianamine-like metallophores are crucial for many pathogens to acquire the transition metals from hosts during invasion. CntL from Staphylococcus aureus (SaCntL) catalyzes the condensation of the 2-aminobutyrate (Ab) moiety of S-adenosylmethionine (SAM) with D-histidine in the biosynthesis of StP. Here, we report the crystal structures of SaCntL in complex with either SAM or two products. The structure of SaCntL consists of an N-terminal four-helix bundle (holding catalytic residue E84) and a C-terminal Rossmann fold (binding the substrates). The sequence connecting the N- and C-terminal domains (N-C linker) in SaCntL was found to undergo conformational alternation between open and closed states. Our structural and biochemical analyses suggested that this intrinsically dynamic interdomain linker forms an additional structural module that plays essential roles in ligand diffusion, recognition, and catalysis. We confirmed that SaCntL stereoselectively carries out the catalysis of D-His but not its enantiomer, L-His, and we found that the N-C linker and active site of SaCntL could accommodate both enantiomers. SaCntL is likely able to bind L-His without catalysis, and as a result, L-His could show inhibitory effects toward SaCntL. These findings provide critical structural and mechanistic insights into CntL, which facilitates a better understanding of the biosynthesis of nicotianamine-like metallophores and the discovery of inhibitors of this process. [ABSTRACT FROM AUTHOR]

Aerobic anoxygenic phototrophs have been isolated from a rich variety of environments including marine ecosystems, freshwater and meromictic lakes, hypersaline springs, and biological soil crusts, all in the hopes of understanding their ecological niche. Over 100 isolates were chosen for this study, representing 44 species from 27 genera. Interactions with Fe3+ and other metal(loid) cations such as Mg2+, V3+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Se4+ and Te2+ were tested using a chromeazurol S assay to detect siderophore or metallophore production, respectively. Representatives from 20 species in 14 genera of α-Proteobacteria, or 30% of strains, produced highly diffusible siderophores that could bind one or more metal(loid)s, with activity strength as follows: Fe > Zn > V > Te > Cu > Mn > Mg > Se > Ni > Co. In addition, γ-proteobacterial Chromocurvus halotolerans, strain EG19 excreted a brown compound into growth medium, which was purified and confirmed to act as a siderophore. It had an approximate size of ~341 Da and drew similarities to the siderophore rhodotorulic acid, a member of the hydroxamate group, previously found only among yeasts. This study is the first to discover siderophore production to be widespread among the aerobic anoxygenic phototrophs, which may be another key method of metal(loid) chelation and potential detoxification within their environments. [ABSTRACT FROM AUTHOR]

To guarantee the supply of critical elements in the future, the development of new technologies is essential. Siderophores have high potential in the recovery and recycling of valuable metals due to their metal-chelating properties. Using the Chrome azurol S assay, 75 bacterial strains were screened to obtain a high-yield siderophore with the ability to complex valuable critical metal ions. The siderophore production of the four selected strains Nocardioides simplex 3E, Pseudomonas chlororaphis DSM 50083, Variovorax paradoxus EPS, and Rhodococcus erythropolis B7g was optimized, resulting in significantly increased siderophore production of N. simplex and R. erythropolis. Produced siderophore amounts and velocities were highly dependent on the carbon source. The genomes of N. simplex and P. chlororaphis were sequenced. Bioinformatical analyses revealed the occurrence of an achromobactin and a pyoverdine gene cluster in P. chlororaphis, a heterobactin and a requichelin gene cluster in R. erythropolis, and a desferrioxamine gene cluster in N. simplex. Finally, the results of the previous metal-binding screening were validated by a proof-of-concept development for the recovery of metal ions from aqueous solutions utilizing C18 columns functionalized with siderophores. We demonstrated the recovery of the critical metal ions V(III), Ga(III), and In(III) from mixed metal solutions with immobilized siderophores of N. simplex and R. erythropolis. [ABSTRACT FROM AUTHOR]