Identification and characterization of a small-molecule metallophore involved in lanthanide metabolism.

Many bacteria secrete metallophores, low-molecular-weight organic compounds that bind ions with high selectivity and affinity, in order to access essential metals from the environment. Previous work has elucidated the structures and biosynthetic machinery of metallophores specific for iron, zinc, nickel, molybdenum, and copper. No physiologically relevant lanthanide-binding metallophore has been discovered despite the knowledge that lanthanide metals (Ln) have been revealed to be essential cofactors for certain alcohol dehydrogenases across a diverse range of phyla. Here, we report the biosynthetic machinery, the structure, and the physiological relevance of a lanthanophore, methylolanthanin. The structure of methylolanthanin exhibits a unique 4-hydroxybenzoate moiety which has not previously been described in other metallophores. We find that production of methylolanthanin is required for normal levels of Ln accumulation in the methylotrophic bacterium Methylobacterium extorquens AM1, while overexpression of the molecule greatly increases bioaccumulation and adsorption. Our results provide a clearer understanding of how Ln-utilizing bacteria sense, scavenge, and store Ln; essential processes in the environment where Ln are poorly bioavailable. More broadly, the identification of this lanthanophore opens doors for study of how biosynthetic gene clusters are repurposed for additional functions and the complex relationship between metal homeostasis and fitness.

Towards new antibiotics classes targeting bacterial metallophores.

The increasing bacterial resistance caused by antibiotic overuse has promoted the search for new antimicrobial strategies. Metals uptake via bacterial metallophores are studied to develop new therapeutics against infectious diseases, because metal ions are essential for bacterial growth and virulence. Metal ions assimilation is mainly dependent on metallophores production which are metal chelators synthetized and produced by bacteria to facilitate metals uptake and are vital for bacterial pathogenicity. Here we highlight the perspective for antimicrobial and therapeutic potential of metallophores through several approaches for metallophores application in antimicrobial therapy.

Unveiling the Pool of Metallophores in Native Environments and Correlation with Their Potential Producers.

For many organisms, metallophores are essential biogenic ligands that ensure metal scavenging and acquisition from their environment. Their identification is challenging in highly organic matter rich environments like peatlands due to low solubilization and metal scarcity and high matrix complexity. In contrast to common approaches based on sample modification by spiking of metal isotope tags, we have developed a two-dimensional (2D) Solid-phase extraction-Liquid chromatography-mass spectrometry (SPE-LC-MS) approach for the highly sensitive (LOD 40 fmol per g of soil), high-resolution direct detection and identification of metallophores in both their noncomplexed (apo) and metal-complexed forms in native environments. The characterization of peat collected in the Bernadouze (France) peatland resulted in the identification of 53 metallophores by a database mass-based search, 36 among which are bacterial. Furthermore, the detection of the characteristic (natural) metal isotope patterns in MS resulted in the detection of both Fe and Cu potential complexes. A taxonomic-based inference method was implemented based on literature and public database (antiSMASH database version 3.0) searches, enabling to associate over 40% of the identified bacterial metallophores with potential producers. In some cases, low completeness with the MIBiG reference BCG might be indicative of alternative producers in the ecosystem. Thus, coupling of metallophore detection and producers' inference could pave a new way to investigate poorly documented environment searching for new metallophores and their producers yet unknown.

CH vs. HC-Promiscuous Metal Sponges in Antimicrobial Peptides and Metallophores.

Histidine and cysteine residues, with their imidazole and thiol moieties that deprotonate at approximately physiological pH values, are primary binding sites for Zn(II), Ni(II) and Fe(II) ions and are thus ubiquitous both in peptidic metallophores and in antimicrobial peptides that may use nutritional immunity as a way to limit pathogenicity during infection. We focus on metal complex solution equilibria of model sequences encompassing Cys-His and His-Cys motifs, showing that the position of histidine and cysteine residues in the sequence has a crucial impact on its coordination properties. CH and HC motifs occur as many as 411 times in the antimicrobial peptide database, while similar CC and HH regions are found 348 and 94 times, respectively. Complex stabilities increase in the series Fe(II) < Ni(II) < Zn(II), with Zn(II) complexes dominating at physiological pH, and Ni(II) ones-above pH 9. The stabilities of Zn(II) complexes with Ac-ACHA-NH2 and Ac-AHCA-NH2 are comparable, and a similar tendency is observed for Fe(II), while in the case of Ni(II), the order of Cys and His does matter-complexes in which the metal is anchored on the third Cys (Ac-AHCA-NH2) are thermodynamically stronger than those where Cys is in position two (Ac-ACHA-NH2) at basic pH, at which point amides start to take part in the binding. Cysteine residues are much better Zn(II)-anchoring sites than histidines; Zn(II) clearly prefers the Cys-Cys type of ligands to Cys-His and His-Cys ones. In the case of His- and Cys-containing peptides, non-binding residues may have an impact on the stability of Ni(II) complexes, most likely protecting the central Ni(II) atom from interacting with solvent molecules.

Metallophores production by bacteria isolated from heavy metal-contaminated soil and sediment at Lerma-Chapala Basin.

Environmental pollution as a result of heavy metals (HMs) is a worldwide problem and the implementation of eco-friendly remediation technologies is thus required. Metallophores, low molecular weight compounds, could have important biotechnological applications in the fields of agriculture, medicine, and bioremediation. This study aimed to isolate HM-resistant bacteria from soils and sediments of the Lerma-Chapala Basin and evaluated their abilities to produce metallophores and to promote plant growth. Bacteria from the Lerma-Chapala Basin produced metallophores for all the tested metal ions, presented a greater production of As3+ metallophores, and showed high HM resistance especially to Zn2+, As5+, and Ni2+. A total of 320 bacteria were isolated with 170 strains showing siderophores synthesis. Members of the Delftia and Pseudomonas genera showed above 92 percent siderophore units (psu) during siderophores production and hydroxamate proved to be the most common functional group among the analyzed siderophores. Our results provided evidence that Lerma-Chapala Basin bacteria and their metallophores could potentially be employed in bioremediation processes or may even have potential for applications in other biotechnological fields.

Functional characterization of culturable fungi from microbiomes of the "conical cobs" Mexican maize (Zea mays L.) landrace.

Mexican maize landraces, produced for local consumption, are adapted to different environmental conditions, and their yield is affected by abiotic and biotic factors, including the use of agrochemicals. The search for sustainable alternatives to agrochemicals includes the study of the culturable microbial communities. In this study, the fungal communities associated with 2 Mexican maize landraces reddish and bluish "conical cobs" were found to be comprised of Ascomycota fungi, represented by 89 strains within 6 orders (Pleosporales, Hypocreales, Onygenales, Capnodiales, Helotiales, and Eurotiales) and 16 genera. Cellulases and metallophores production were the primary enzymatic products and plant growth-promoting activities were detected among the isolates. Penicillium, Didymella, and Fusarium strains had the most active enzymatic and plant growth promoting activities, however, Aspergillus sp. HES2-2.2, Talaromyces sp. RS1-7, and Penicillium sp. HFS3-3 showed antagonistic activity against the four phytopathogenic Fusarium strains Fusarium oxysporum, Fusarium sambucinum, Fusarium fujikuroi and Fusarium incarnatum-equiseti and also a high and diverse production of enzymatic and plant growth promoting activities; here we identified fungal strains as candidates to promote maize growth.

Multiple siderophores: bug or feature?

It is common for bacteria to produce chemically diverse sets of small Fe-binding molecules called siderophores. Studies of siderophore bioinorganic chemistry have firmly established the role of these molecules in Fe uptake and provided great insight into Fe complexation. However, we still do not fully understand why microbes make so many siderophores. In many cases, the release of small structural variants or siderophore fragments has been ignored, or considered as an inefficiency of siderophore biosynthesis. Yet, in natural settings, microbes live in complex consortia and it has become increasingly clear that the secondary metabolite repertoires of microbes reflect this dynamic environment. Multiple siderophore production may, therefore, provide a window into microbial life in the wild. This minireview focuses on three biochemical routes by which multiple siderophores can be released by the same organism-multiple biosynthetic gene clusters, fragment release, and precursor-directed biosynthesis-and highlights emergent themes related to each. We also emphasize the plurality of reasons for multiple siderophore production, which include enhanced iron uptake via synergistic siderophore use, microbial warfare and cooperation, and non-classical functions such as the use of siderophores to take up metals other than Fe.

Bacterial Strategies to Maintain Zinc Metallostasis at the Host-Pathogen Interface.

Among the biologically required first row, late d-block metals from MnII to ZnII, the catalytic and structural reach of ZnII ensures that this essential micronutrient touches nearly every major metabolic process or pathway in the cell. Zn is also toxic in excess, primarily because it is a highly competitive divalent metal and will displace more weakly bound transition metals in the active sites of metalloenzymes if left unregulated. The vertebrate innate immune system uses several strategies to exploit this "Achilles heel" of microbial physiology, but bacterial evolution has responded in kind. This review highlights recent insights into transcriptional, transport, and trafficking mechanisms that pathogens use to "win the fight" over zinc and thrive in an otherwise hostile environment.

Solution studies, synthesis and antibacterial activity of Ga(III) complexes with bis-kojate derivatives.

Given the recognized major problem of microbial drug resistance for human health, new metal-based drugs have been currently explored for their antimicrobial properties, including gallium-based compounds as potential metallophores that could perturb Fe's interactions with proteins. Herein we have designed and synthesized two bis-kojate ligands (named L4 and L6) and studied their Ga(III) complexes for their physico-chemical and biological properties. In particular a detailed study of their complexation properties in aqueous solution, showed equilibrium models with formation of quite stable dinuclear 2:3 metal:ligand complexes, though with different stability. Solid state complexes were also prepared and characterized and complementary DFT studies indicated that [Ga2(L4)3] complex, with higher stability, seems to adopt a three-ligand bridging conformation, while that for L6 adopt a one ligand bridging conformation. Preliminary investigation of the antibacterial activity of these gallium complexes showed antipseudomonal activity, which appeared higher for the complex with L4, a feature of potential interest for the scientific community.

Predicting metallophore structure and function through genome mining.

Metallophores are small molecule chelators that many microbes use to obtain trace metals from their environment. Through genome mining, where genomes are scanned for metallophore biosynthesis genes, one can not only identify which organisms are likely to produce a metallophore, but also predict the metallophore structure, thus preventing undesired reisolation of known compounds and accelerating characterization. Furthermore, the presence of accessory genes for the transport, utilization, and regulation can suggest the biological function and fate of a metallophore. Modern, user-friendly tools have made powerful genomic analyses accessible to scientists with no bioinformatics experience, but these tools are often not utilized to their full potential. This chapter provides an introduction to metallophore genomics and demonstrates how to use the free, publicly available antiSMASH platform to infer metallophore function and structure.

Overview of Yersinia pestis Metallophores: Yersiniabactin and Yersinopine.

The pathogenic anaerobic bacteria Yersinia pestis (Y. pestis), which is well known as the plague causative agent, has the ability to escape or inhibit innate immune system responses, which can result in host death even before the activation of adaptive responses. Bites from infected fleas in nature transmit Y. pestis between mammalian hosts causing bubonic plague. It was recognized that a host's ability to retain iron is essential in fighting invading pathogens. To proliferate during infection, Y. pestis, like most bacteria, has various iron transporters that enable it to acquire iron from its hosts. The siderophore-dependent iron transport system was found to be crucial for the pathogenesis of this bacterium. Siderophores are low-molecular-weight metabolites with a high affinity for Fe3+. These compounds are produced in the surrounding environment to chelate iron. The siderophore secreted by Y. pestis is yersiniabactin (Ybt). Another metallophore produced by this bacterium, yersinopine, is of the opine type and shows similarities with both staphylopine and pseudopaline produced by Staphylococcus aureus and Pseudomonas aeruginosa, respectively. This paper sheds light on the most important aspects of the two Y. pestis metallophores as well as aerobactin a siderophore no longer secreted by this bacterium due to frameshift mutation in its genome.

New mescaline-related N-acylhydrazone and its unsubstituted benzoyl derivative: Promising metallophores for copper-associated deleterious effects relief in Alzheimer's disease.

Alzheimer's disease (AD) is related to the presence of extracellular aggregated amyloid-ß peptide (Aß), which binds copper(II) with high affinity in its N-terminal region. In this sense, two new 1-methylimidazole-containing N-acylhydrazonic metallophores, namely, X1TMP and X1Benz, were synthesized as hydrochlorides and characterized. The compound X1TMP contains the 3,4,5-trimethoxybenzoyl moiety present in the structure of mescaline, a natural hallucinogenic protoalkaloid that occurs in some species of cacti. Single crystals of X1Benz, the unsubstituted derivative of X1TMP, were obtained. The experimental partition coefficients of both compounds were determined, as well as their apparent affinity for Cu2+ in aqueous solution. Ascorbate consumption assays showed that these N-acylhydrazones are able to lessen the production of ROS by the Cu(Aß)-system, and a short-time scale aggregation study, measured through turbidity and confirmed by TEM images, revealed their capacity in preventing Aß fibrillation at equimolar conditions in the presence and absence of copper. 1H15N HSQC NMR experiments demonstrated a direct interaction between Aß and X1Benz, the most soluble of the compounds. The Cu2+ sequestering potential of this hydrazone towards Aß was explored by 1H NMR. Although increasing amounts of X1Benz were unexpectedly not efficient at removing the metal-induced perturbations in Aß backbone amides, the broadening effects observed on the compound's signals indicate the formation of a ternary Aß­copper-X1Benz species, which can be responsible for the observed ROS-lessening and aggregation-preventing activities. Overall, the N-acylhydrazones X1TMP and X1Benz have shown promising prospects as agents for the treatment of AD.

Isoavenaciol and 7-hydroxy-isoavenaciol: Zn-chelating metallophores produced by root-endophytic Pezicula ericae in a Zn-accumulating plant, Aucuba japonica.

Metallophores are low-molecular-weight compounds capable of chelating heavy metals, which have recently been reported to alleviate heavy metal stress in plants. We isolated two undescribed compounds as Zn-chelating metallophores from the culture broth of the root endophytic Pezicula ericae w12-25, which was collected from a Zn-accumulating plant, Aucuba japonica Thunb. These two compounds were determined to be (3aS,4S,6aR)-3a-hydroxy-3-methylene-4-octyldihydrofuro[3,4-b]furan-2,6(3H,4H)-dione and (3S,3aS,4S,6aR)-3a-hydroxy-3-(hydroxymethyl)-4-octyldihydrofuro[3,4-b]furan-2,6(3H,4H)-dione using spectroscopic methods (HRMS, 1H and 13C NMR, and 2D NMR) and X-ray crystallography, respectively. The two compounds, classified as furofurandiones, were named isoavenaciol and 7-hydroxy-isoavenaciol. After the hydrolysis of the lactone moiety, isoavenaciol would release the carboxyl group to show Zn-chelating activity. Their antifungal activities were confirmed using Cladosporium herbarum (AHU9262).

The Key Element Role of Metallophores in the Pathogenicity and Virulence of Staphylococcus aureus: A Review.

The ubiquitous bacterium Staphylococcus aureus causes many diseases that sometimes can be fatal due to its high pathogenicity. The latter is caused by the ability of this pathogen to secrete secondary metabolites, enabling it to colonize inside the host causing infection through various processes. Metallophores are secondary metabolites that enable bacteria to sequester metal ions from the surrounding environment since the availability of metal ions is crucial for bacterial metabolism and virulence. The uptake of iron and other metal ions such as nickel and zinc is one of these essential mechanisms that gives this germ its virulence properties and allow it to overcome the host immune system. Additionally, extensive interactions occur between this pathogen and other bacteria as they compete for resources. Staphylococcus aureus has high-affinity metal import pathways including metal ions acquisition, recruitment and metal-chelate complex import. These characteristics give this bacterium the ability to intake metallophores synthesized by other bacteria, thus enabling it to compete with other microorganisms for the limited nutrients. In scarce host conditions, free metal ions are extremely low because they are confined to storage and metabolic molecules, so metal ions are sequestered by metallophores produced by this bacterium. Both siderophores (iron chelating molecules) and staphylopine (wide- spectrum metallophore) are secreted by Staphylococcus aureus giving it infectious properties. The genetic regulation of the synthesis and export together with the import of metal loaded metallophores are well established and are all covered in this review.

A Review of Pseudomonas aeruginosa Metallophores: Pyoverdine, Pyochelin and Pseudopaline.

P. aeruginosa is a common Gram-negative bacterium found in nature that causes severe infections in humans. As a result of its natural resistance to antibiotics and the ability of biofilm formation, the infection with this pathogen can be therapeutic challenging. During infection, P. aeruginosa produces secondary metabolites such as metallophores that play an important role in their virulence. Metallophores are metal ions chelating molecules secreted by bacteria, thus allowing them to survive in the host under metal scarce conditions. Pyoverdine, pyochelin and pseudopaline are the three metallophores secreted by P. aeruginosa. Pyoverdines are the primary siderophores that acquire iron from the surrounding medium. These molecules scavenge and transport iron to the bacterium intracellular compartment. Pyochelin is another siderophore produced by this bacterium, but in lower quantities and its affinity for iron is less than that of pyoverdine. The third metallophore, pseudopaline, is an opine narrow spectrum ion chelator that enables P. aeruginosa to uptake zinc in particular but can transport nickel and cobalt as well. This review describes all the aspects related to these three metallophore, including their main features, biosynthesis process, secretion and uptake when loaded by metals, in addition to the genetic regulation responsible for their synthesis and secretion.

Zinc-binding metallophores protect Pseudomonas aeruginosa from calprotectin-mediated metal starvation.

Pseudomonas aeruginosa is known to exhibit considerable resistance to the antimicrobial activity of the metal-sequestering protein calprotectin (CP). In this study, we demonstrate that although CP induces zinc deficiency in P. aeruginosa, a strain unable to import zinc through the two most important metal acquisition systems, namely ZnuABC and ZrmABCD, maintains significant growth capacity in the presence of high concentrations of CP. Furthermore, we have shown that nicotianamine, a molecule structurally similar to the metallophore pseudopaline, can favor the acquisition of the metal even in the presence of CP. To gain insights into the mechanisms through which metallophores can promote zinc acquisition, we analyzed the effect of nicotianamine on the activity of the metallo-ß-lactamase VIM-1. Our data suggest that metallophores released by bacteria in response to zinc deficiency can extract the protein-bound metal. The ability to interfere with the binding of metals to proteins, as well as favoring the acquisition of zinc, may contribute to increasing the resistance of P. aeruginosa to the antimicrobial action of CP.

Secondary Metabolites Governing Microbiome Interaction of Staphylococcal Pathogens and Commensals.

Various Staphylococcus species colonize skin and upper airways of warm-blooded animals. They compete successfully with many other microorganisms under the hostile and nutrient-poor conditions of these habitats using mechanisms that we are only beginning to appreciate. Small-molecule mediators, whose biosynthesis requires complex enzymatic cascades, so-called secondary metabolites, have emerged as crucial components of staphylococcal microbiome interactions. Such mediators belong to a large variety of compound classes and several of them have attractive properties for future drug development. They include, for instance, bacteriocins such as lanthipeptides, thiopeptides, and fibupeptides that inhibit bacterial competitor species; signaling molecules such as thiolactone peptides that induce or inhibit sensory cascades in other bacteria; or metallophores such as staphyloferrins and staphylopine that scavenge scant transition metal ions. For some secondary metabolites such as the aureusimines, the exact function remains to be elucidated. How secondary metabolites shape the fitness of Staphylococcus species in the complex context of other microbial and host defense factors remains a challenging field of future research. A detailed understanding will help to harness staphylococcal secondary metabolites for excluding the pathogenic species Staphylococcus aureus from the nasal microbiomes of at-risk patients, and it will be instrumental for the development of advanced anti-infective interventions.

Secondary metabolites released by the rhizosphere bacteria Arthrobacter oxydans and Kocuria rosea enhance plant availability and soil-plant transfer of germanium (Ge) and rare earth elements (REEs).

Here, we explore effects of metallophore-producing rhizobacteria on the plant availability of germanium (Ge) and rare earth elements (REEs). Five isolates of the four species Rhodococcus erythropolis, Arthrobacter oxydans, Kocuria rosea and Chryseobacterium koreense were characterized regarding their production of element-chelators using genome-mining, LC-MS/MS analysis and solid CAS-assay. Additionally, a soil elution experiment was conducted in order to identify isolates that increase solubility of Ge and REEs in soil solution. A. oxydans ATW2 and K. rosea ATW4 released desferrioxamine-, bacillibactin- and surfactin-like compounds that mobilized Ge and REEs as well as P, Fe, Si and Ca in soil. Subsequently, oat, rapeseed and reed canary grass were cultivated on soil and sand and treated with cells and iron depleted culture supernatants of A. oxydans ATW2 and K. rosea ATW4. Inoculation increased plant yield and shoot phosphorus (P), manganese (Mn), Ge and REE concentrations. However, effects of the inoculation varied substantially between the growth substrates and plant species. On sand, A. oxydans ATW2 increased accumulation of REEs in all plant species and root-shoot translocation in rapeseed, while K. rosea ATW4 enhanced REE accumulation in rapeseed only, without effects on other plant species. Sand-cultured oat plants showed increased Ge accumulation and root-shoot translocation in presence of A. oxydans ATW2 cells and K. rosea ATW4 supernatant; however, there was no effect on other plant species, irrespective the growth substrate used. In contrast, soil-cultured rapeseed showed enhanced REE accumulation in presence of cells of A. oxydans ATW2 while there were no effects on other plant species and Ge. The processes involved are not yet fully understood. Nevertheless, we demonstrated that chemical microbe-soil-plant relationships influence plant availability of nutrients together with Ge and REEs, which has major implications on our understanding of biogeochemical element cycling and development of sustainable bioremediation and biomining technologies.

The biochemistry of lanthanide acquisition, trafficking, and utilization.

Lanthanides are relative newcomers to the field of cell biology of metals; their specific incorporation into enzymes was only demonstrated in 2011, with the isolation of a bacterial lanthanide- and pyrroloquinoline quinone-dependent methanol dehydrogenase. Since that discovery, the efforts of many investigators have revealed that lanthanide utilization is widespread in environmentally important bacteria, and parallel efforts have focused on elucidating the molecular details involved in selective recognition and utilization of these metals. In this review, we discuss the particular chemical challenges and advantages associated with biology's use of lanthanides, as well as the currently known lanthano-enzymes and -proteins (the lanthanome). We also review the emerging understanding of the coordination chemistry and biology of lanthanide acquisition, trafficking, and regulatory pathways. These studies have revealed significant parallels with pathways for utilization of other metals in biology. Finally, we discuss some of the many unresolved questions in this burgeoning field and their potentially far-reaching applications.

Micromonospora metallophores: A plant growth promotion trait useful for bacterial-assisted phytoremediation?

Heavy metal pollution in the environment is an increasing problem due to natural and anthropogenic activities. The use of bacteria for bioremediation of soils contaminated with heavy metals has gained a lot of attention as it can be considered effective, economic and environmentally sustainable. In this work, we investigated the capacity of endophytic Micromonospora strains isolated from different legumes, to produce metallophores against a variety of heavy metals in vitro. Genome mining using available endophytic Micromonospora genome sequences revealed the presence of genes related to metal acquisition, iron metabolism and resistance to toxic compounds. In vitro production of metallophores demonstrated that all strains tested produced chelates against arsenic, cobalt, copper, chromium, iron, mercury, molybdenum, nickel, vanadium and zinc in different amounts. In addition, the plant growth promotion effect of strains GAR05 and PSN13 on Arabidopsis thaliana grown in the presence of several heavy metals was tested. Under these conditions, the plants inoculated with the strain GAR05 showed significant growth when compared to the control plants suggesting a plant growth promotion effect in the form of tolerance to the toxic substances. Furthermore, during this plant-bacterium interaction, a new bacterial structure named root-bead was observed on the roots of A. thaliana suggesting a strong interaction between the two organisms and a clear positive effect of the bacterium on the plant. Overall, these results highlight the potential use of endophytic Micromonospora strains for bacterial-assisted phytoremediation of contaminated sites.