RESUMO
Microorganisms, plants, and animals alike have specialized acquisition pathways for obtaining metals, with microorganisms and plants biosynthesizing and secreting small molecule natural products called siderophores and metallophores with high affinities and specificities for iron or other non-iron metals, respectively. This chapter details a novel approach to discovering metal-binding molecules, including siderophores and metallophores, from complex samples ranging from microbial supernatants to biological tissue to environmental samples. This approach, called Native Metabolomics, is a mass spectrometry method in which pH adjustment and metal infusion post-liquid chromatography are interfaced with ion identity molecular networking (IIMN). This rule-based data analysis workflow that enables the identification of metal-binding species based on defined mass (m/z) offsets with the same chromatographic profiles and retention times. Ion identity molecular networking connects compounds that are structurally similar by their fragmentation pattern and species that are ion adducts of the same compound by chromatographic shape correlations. This approach has previously revealed new insights into metal binding metabolites, including that yersiniabactin can act as a biological zincophore (in addition to its known role as a siderophore), that the recently elucidated lepotchelin natural products are cyanobacterial metallophores, and that antioxidants in traditional medicine bind iron. Native metabolomics can be conducted on any liquid chromatography-mass spectrometry system to explore the binding of any metal or multiple metals simultaneously, underscoring the potential for this method to become an essential strategy for elucidating biological metal-binding molecules.
Assuntos
Espectrometria de Massas , Metabolômica , Sideróforos , Sideróforos/metabolismo , Sideróforos/química , Sideróforos/análise , Metabolômica/métodos , Espectrometria de Massas/métodos , Cromatografia Líquida/métodos , Ferro/metabolismo , Ferro/análiseRESUMO
Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MSI) is a label-free technique, producing images where pixels contain mass spectra. The technique allows the visualization of the spatial distribution of (bio)molecules from metabolites to proteins, on surfaces such as tissues sections or bacteria culture media. One particularly exciting example of MALDI-MSI use rests on its potential to localize ionized compounds produced during microbial interactions and chemical communication, offering a molecular snapshot of metabolomes at a given time. The huge size and the complexity of generated MSI data make the processing of the data challenging, which requires the use of computational methods. Despite recent advances, currently available commercial software relies mainly on statistical tools to identify patterns, similarities, and differences within data sets. However, grouping m/z values unique to a given data set according to microbiological contexts, such as coculture experiments, still requires tedious manual analysis. Here we propose a nontargeted method exploiting the differential signals between negative controls and tested experimental conditions, i.e., differential signal filtering (DSF), and a scoring of the ion images using image structure filtering (ISF) coupled with a fold change score between the controls and the conditions of interest. These methods were first applied to coculture experiments involving Escherichia coli and Streptomyces coelicolor, revealing specific MS signals during bacterial interaction. Two case studies were also investigated: (i) cellobiose-mediated induction for the pathogenicity of Streptomyces scabiei, the causative agent of common scab on root and tuber crops, and (ii) iron-repressed production of siderophores of S. scabiei. This report proposes guidelines for MALDI-MSI data treatment applied in the case of microbiology contexts, with enhanced ion peak annotation in specific culture conditions. The strengths and weaknesses of the methods are discussed.
Assuntos
Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Processamento de Imagem Assistida por Computador/métodos , Escherichia coli/química , Escherichia coli/metabolismo , Metaboloma/fisiologia , Sideróforos/análise , Sideróforos/metabolismo , Sideróforos/química , Bactérias/metabolismo , Bactérias/químicaRESUMO
Research on harmful algal blooms has focused on macronutrients, yet recent research increasingly indicates that understanding micronutrient roles is also important in the development of effective environmental management interventions. Here, we report results on metallophore production from mesocosms amended with copper and iron (enzymatic co-factors in photosynthetic electron transport) to probe questions of how cyanobacteria navigate the divide between copper nutrition, copper toxicity, and issues with iron bioavailability. These experiments utilized Microcystis, Chlorella and Desmodesmus spp., in mono- and mixed-cultures in lake water from a large, hypereutrophic lake (Taihu, China). To initiate experiments, copper and iron amendments were added to mesocosms containing algae that had been acclimated to achieve a state of copper and iron limitation. Mesocosms were analyzed over time for a range of analytes including algal growth parameters, algal assemblage progression, copper/iron concentrations and biomolecule production of chalkophore, siderophore and total microcystins. Community Trajectory Analysis and other multivariate methods were used for analysis resulting in our findings: 1) Microcystis spp. manage copper/iron requirements though a dynamically phased behavior of chalkophore/siderophore production according to their copper and iron limitation status (chalkophore correlates with Cu concentration, R2 = 0.99, and siderophore correlates with the sum of Cu and Fe concentrations, R2 = 0.98). 2) A strong correlation was observed between the production of chalkophore and the cyanobacterial toxin microcystin (R2 = 0.76)-Chalkophore is a predictor of microcystin production. 3) Based on our results and literature, we posit that Microcystis spp. produces microcystin in response to copper/iron availability to manage photosystem productivity and effect an energy-saving status. Results from this work underscore the importance of micronutrients in influencing harmful algal bloom progression and represents a major advance in understanding the ecological function for the cyanobacterial toxin microcystin as a hallmark of micronutrient limitation stress.
Assuntos
Chlorella , Microcystis , Proliferação Nociva de Algas , Cobre/análise , Microcistinas/análise , Ferro/análise , Sideróforos/análise , Lagos/microbiologia , Micronutrientes/análiseRESUMO
As one of the most accumulative toxic heavy metals, cadmium (Cd) poses a major threat to human health. Bacterial siderophores, as small molecules with metal-absorbing ability, have great potential activity for Cd-reduction. In this study, the siderophore-producing bacterialstrain FCH-CR2 was isolated from a high-Cd contaminated soil using the CAS method. Leclercia adecarboxylata was identified through 16S rRNA sequence, homology analysis, colony morphology, physiological and biochemical tests. A siderophore, catechol type 2,3-dihydroxy-N-benzoyl-l-serine (DHBS) secreted by FCH-CR2, was purified using RP-HPLC and identified by LC-MS/MS. Intraperitoneal injection of DHBS significantly increased fecal Cd levels, and reduced Cd accumulation in organs. In density flooding theory (DFT) analysis, DHBS may bind to Cd via the hydroxyl site on the benzene ring. Besides, the isothermal titration calorimetry (ITC) assay revealed that the formation of Cd-DHBS is a spontaneous and endothermic reaction with ΔG = -21.4 kJ/mol and ΔH = 1.51 ± 0.142 kJ/mol.
Assuntos
Metais Pesados , Poluentes do Solo , Humanos , Sideróforos/análise , Sideróforos/metabolismo , Cádmio/análise , RNA Ribossômico 16S/genética , Cromatografia Líquida , Espectrometria de Massas em Tandem , Quelantes , Poluentes do Solo/análise , Solo/química , Metais Pesados/análiseRESUMO
Dietary selenium (Se) is an effective strategy to meet Se requirement of human body, and Se biofortification in crops in seleniferous soils with selenobacteria represents an eco-friendly biotechnique. In this study, we tested the effectiveness of siderophore-producing bacterial (SPB) synthetic communities (SynComs) in promoting plant Se uptake in a subtropical seleniferous soil where the fixation of Se by ferric-oxides is severe. The results indicated that SPB SynComs drastically elevated soil bioavailable Se content by up to 68.7 %, and significantly increased plant Se concentration and uptake by up to 83.1 % and 92.2 %, respectively. Seven out of ten SPB isolates in the SynComs were enriched in soils after 120 days of inoculation. Additionally, variation partitioning analysis (VPA) revealed that the contribution of soil bacterial community (up to 42.8 %) to the increased plant Se uptake was much greater than that of soil bioavailable Se (up to 5.1 %), suggesting a direct pathway other than the pathway of mobilizing Se. The relative abundances of some operational taxonomic units (OTUs) showed significantly positive relationship with plant Se status but not with soil Se status, which supports the results of VPA. Network analysis indicates that some inoculated SPB isolates promoted plant Se uptake by regulating the native bacterial taxa. Taken together, this study demonstrates that SPB can be used in Se biofortification in crops, especially in subtropical soils.
Assuntos
Microbiota , Selênio , Poluentes do Solo , Humanos , Solo , Selênio/análise , Sideróforos/análise , Sideróforos/metabolismo , Disponibilidade Biológica , Produtos Agrícolas/metabolismo , Bactérias/metabolismo , Poluentes do Solo/análiseRESUMO
Siderophores are iron-chelating molecules that solubilize Fe3+ for microbial utilization and facilitate colonization or infection of eukaryotes by liberating host iron for bacterial uptake. By fluorescently labeling membrane receptors and binding proteins, we created 20 sensors that detect, discriminate, and quantify apo- and ferric siderophores. The sensor proteins originated from TonB-dependent ligand-gated porins (LGPs) of Escherichia coli (Fiu, FepA, Cir, FhuA, IutA, BtuB), Klebsiella pneumoniae (IroN, FepA, FyuA), Acinetobacter baumannii (PiuA, FepA, PirA, BauA), Pseudomonas aeruginosa (FepA, FpvA), and Caulobacter crescentus (HutA) from a periplasmic E. coli binding protein (FepB) and from a human serum binding protein (siderocalin). They detected ferric catecholates (enterobactin, degraded enterobactin, glucosylated enterobactin, dihydroxybenzoate, dihydroxybenzoyl serine, cefidericol, MB-1), ferric hydroxamates (ferrichromes, aerobactin), mixed iron complexes (yersiniabactin, acinetobactin, pyoverdine), and porphyrins (hemin, vitamin B12). The sensors defined the specificities and corresponding affinities of the LGPs and binding proteins and monitored ferric siderophore and porphyrin transport by microbial pathogens. We also quantified, for the first time, broad recognition of diverse ferric complexes by some LGPs, as well as monospecificity for a single metal chelate by others. In addition to their primary ferric siderophore ligands, most LGPs bound the corresponding aposiderophore with â¼100-fold lower affinity. These sensors provide insights into ferric siderophore biosynthesis and uptake pathways in free-living, commensal, and pathogenic Gram-negative bacteria.
Assuntos
Proteínas de Bactérias , Corantes Fluorescentes , Bactérias Gram-Negativas Quimiolitotróficas , Sideróforos , Acinetobacter baumannii , Proteínas da Membrana Bacteriana Externa/metabolismo , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Caulobacter crescentus , Enterobactina/análise , Enterobactina/metabolismo , Escherichia coli/metabolismo , Corantes Fluorescentes/química , Bactérias Gram-Negativas Quimiolitotróficas/química , Bactérias Gram-Negativas Quimiolitotróficas/genética , Bactérias Gram-Negativas Quimiolitotróficas/metabolismo , Humanos , Ferro/metabolismo , Klebsiella pneumoniae , Sideróforos/análise , Sideróforos/metabolismoRESUMO
There is an emerging need for accurate and rapid identification of bacteria in the human body to achieve diverse biomedical objectives. Copper homeostasis is vital for the survival of bacterial species owing to the roles of the metal as a nutrient, respiratory enzyme cofactor, and a toxin. Here, we report the development of a copper-64-labeled bacterial metal chelator, yersiniabactin, to exploit a highly conserved metal acquisition pathway for noninvasive and selective imaging of bacteria. Compared with traditional techniques used to manufacture probes, our strategy simplifies the process considerably by combining the function of metal attachment and cell recognition to the same molecule. We demonstrate, for the first time to our knowledge, how a copper-64 PET probe can be used to identify specific bacterial populations, monitor antibiotic treatment outcomes, and track bacteria in diverse niches in vivo.
Assuntos
Infecções Bacterianas , Cobre/metabolismo , Fenóis , Tomografia por Emissão de Pósitrons/métodos , Sideróforos , Tiazóis , Animais , Bactérias/química , Bactérias/metabolismo , Infecções Bacterianas/diagnóstico por imagem , Infecções Bacterianas/microbiologia , Modelos Animais de Doenças , Ecocardiografia , Feminino , Camundongos , Camundongos Endogâmicos BALB C , Imagem Molecular , Fenóis/análise , Fenóis/química , Fenóis/metabolismo , Sideróforos/análise , Sideróforos/química , Sideróforos/metabolismo , Tiazóis/análise , Tiazóis/química , Tiazóis/metabolismoRESUMO
This study evaluated the phytoextraction capacity of the fern Pteris vittata grown on a natural arsenic-rich soil of volcanic-origin from the Viterbo area in central Italy. This calcareous soil is characterized by an average arsenic concentration of 750 mg kg-1, of which 28% is bioavailable. By means of micro-energy dispersive X-ray fluorescence spectrometry (µ-XRF) we detected As in P. vittata fronds after just 10 days of growth, while a high As concentrations in fronds (5,000 mg kg-1), determined by Inductively coupled plasma-optical emission spectrometry (ICP-OES), was reached after 5.5 months. Sixteen arsenate-tolerant bacterial strains were isolated from the P. vittata rhizosphere, a majority of which belong to the Bacillus genus, and of this majority only two have been previously associated with As. Six bacterial isolates were highly As-resistant (> 100 mM) two of which, homologous to Paenarthrobacter ureafaciens and Beijerinckia fluminensis, produced a high amount of IAA and siderophores and have never been isolated from P. vittata roots. Furthermore, five isolates contained the arsenate reductase gene (arsC). We conclude that P. vittata can efficiently phytoextract As when grown on this natural As-rich soil and a consortium of bacteria, largely different from that usually found in As-polluted soils, has been found in P. vittata rhizosphere.
Assuntos
Arsênio/análise , Beijerinckiaceae/metabolismo , Micrococcaceae/metabolismo , Pteris/química , Solo/química , Arseniato Redutases/genética , Arseniato Redutases/metabolismo , Arsênio/metabolismo , Arsênio/toxicidade , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Beijerinckiaceae/química , Beijerinckiaceae/isolamento & purificação , Biodegradação Ambiental , Farmacorresistência Bacteriana/genética , Micrococcaceae/química , Micrococcaceae/isolamento & purificação , Raízes de Plantas/química , Raízes de Plantas/metabolismo , Raízes de Plantas/microbiologia , Pteris/metabolismo , Pteris/microbiologia , Rizosfera , Sideróforos/análise , Sideróforos/metabolismo , Microbiologia do Solo , Poluentes do Solo/análise , Poluentes do Solo/metabolismo , Espectrofotometria AtômicaRESUMO
Interspecies bacterial competition may occur via cell-associated or secreted determinants and is key to successful niche colonization. We previously evolved Pseudomonas aeruginosa in the presence of Staphylococcus aureus and identified mutations in the Wsp surface-sensing signalling system. Surprisingly, a ΔwspF mutant, characterized by increased c-di-GMP levels and biofilm formation capacity, showed potent killing activity towards S. aureus in its culture supernatant. Here, we used an unbiased metabolomic analysis of culture supernatants to identify rhamnolipids, alkyl quinoline N-oxides and two siderophores as members of four chemical clusters, which were more abundant in the ΔwspF mutant supernatants. Killing activities were quorum-sensing controlled but independent of c-di-GMP levels. Based on the metabolomic analysis, we formulated a synthetic cocktail of four compounds, showing broad-spectrum anti-bacterial killing, including both Gram-positive and Gram-negative bacteria. The combination of quorum-sensing-controlled killing and Wsp-system mediated biofilm formation endows P. aeruginosa with capacities essential for niche establishment and host colonization.
Assuntos
Antibacterianos/metabolismo , Antibiose/fisiologia , Pseudomonas aeruginosa/metabolismo , Staphylococcus aureus/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , GMP Cíclico/análogos & derivados , GMP Cíclico/análise , Glicolipídeos/análise , Oligopeptídeos/análise , Fenóis/análise , Pseudomonas aeruginosa/genética , Quinolinas/análise , Percepção de Quorum/genética , Sideróforos/análise , Staphylococcus aureus/genética , Staphylococcus aureus/metabolismo , Tiazóis/análiseAssuntos
Úlcera da Perna/microbiologia , Imagem Óptica/instrumentação , Infecções por Pseudomonas/diagnóstico , Pseudomonas aeruginosa/metabolismo , Raios Ultravioleta , Idoso , Proteínas de Bactérias/análise , Humanos , Úlcera da Perna/metabolismo , Masculino , Oligopeptídeos/análise , Infecções por Pseudomonas/metabolismo , Pseudomonas aeruginosa/isolamento & purificação , Sideróforos/análiseRESUMO
A Gram-stain-positive, aerobic, spore-forming actinobacterial strain, designated 160415T, was isolated from a surface soil sample, which was formed on basaltic parent material, collected from Samsun, Turkey. Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 160415T clustered closely with species of the genus Nonomuraea, and showed the highest sequence similarity to Nonomuraea zeae NEAU-ND5T, Nonomuraea candida HMC10T and Nonomuraea turkmeniaca DSM 43926T with 99.1%, 98.9% and 98.7%, respectively. Chemotaxonomic properties including major menaquinones, diaminopimelic acid, sugar and phospholipid profiles also confirmed the affiliation of the strain to the genus Nonomuraea. The DNA G+C content of strain 160415T was 69.6 mol%. DNA-DNA hybridization and average nucleotide identity values between the strain and closely related type strains were less than the recommended cut-off values. On the basis of phylogenetic relationships, genotypic and phenotypic characterizations, strain 160415T represents a novel species of the genus Nonomuraea, for which the name Nonomuraea basaltis sp. nov. is proposed. The type strain is 160415T (= KCTC 39875T = DSM 104309T).
Assuntos
Actinobacteria , Actinobacteria/classificação , Actinobacteria/genética , Actinobacteria/isolamento & purificação , Actinomycetales/genética , Técnicas de Tipagem Bacteriana , Composição de Bases/genética , DNA Bacteriano/genética , Ácidos Graxos/análise , Genoma Bacteriano/genética , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Sideróforos/análise , Sideróforos/metabolismo , Solo , Microbiologia do Solo , TurquiaRESUMO
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.
Assuntos
Sideróforos/análise , Staphylococcus aureus/patogenicidade , Abscesso/microbiologia , Animais , Citratos/análise , Interações Hospedeiro-Patógeno , Ferro/metabolismo , Camundongos , Ornitina/análogos & derivados , Ornitina/análise , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Infecções Estafilocócicas/microbiologia , Infecções Estafilocócicas/patologiaRESUMO
Deferoxamine, a metal chelator, has been shown to be neuroprotective in animal models of ischemic stroke, traumatic brain injury and both subarachnoid and intracerebral hemorrhage. Intranasal deferoxamine (IN DFO) has also shown promise as a potential treatment for multiple neurodegenerative diseases, including Parkinson's and Alzheimer's. However, there have been no attempts to thoroughly understand the dynamics and pharmacokinetics of IN DFO. We developed a new high-performance liquid-chromatography electrospray-tandem mass spectrometry (HPLC/ESI-MS2) method to quantify the combined total levels of DFO, ferrioxamine (FO; DFO bound to iron), and aluminoxamine (AO; aluminum-bound DFO) in brain tissue using a custom-synthesized deuterated analogue (DFO-d7, Medical Isotopes Inc., Pelham NH) as an internal standard. We applied our method toward understanding the pharmacokinetics of IN DFO delivery to the brain and blood of rats from 15 min to 4 h after delivery. We found that IN delivery successfully targets DFO to the brain to achieve concentrations of 0.5-15 µM in various brain regions within 15 min, and decreasing though still detectable after 4 h. Systemic exposure was minimized as assessed by concentration in blood serum. Serum concentrations were 0.02 µM at 15 min and no more than 0.1 µM at later time points. Compared to blood serum, brain region-specific drug exposure (as measured by area under the curve) ranged from slightly under 10 times exposure in the hippocampus to almost 200 times exposure in the olfactory bulb with IN DFO delivery. These findings represent a major step toward future method development, pharmacokinetic studies, and clinical trials for this promising therapeutic.
Assuntos
Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Desferroxamina/administração & dosagem , Desferroxamina/metabolismo , Sideróforos/administração & dosagem , Sideróforos/metabolismo , Administração Intranasal , Animais , Química Encefálica/efeitos dos fármacos , Química Encefálica/fisiologia , Desferroxamina/análise , Espectrometria de Massas/métodos , Ratos , Ratos Sprague-Dawley , Sideróforos/análiseRESUMO
Trichodesmium is a globally important marine nitrogen fixing cyanobacteria which forms colonies and utilizes atmospherically derived dust as a source for the limiting micro-nutrient iron. Here we report the identification of metallophores isolated from incubations of natural Trichodesmium colonies collected from the Gulf of Aqaba in the Red Sea. Three of our compounds were identified as the ferrioxamine siderophores B, E, and G. The remaining fifteen metallophores had mass to charge ratios that, to our knowledge, are not common to known siderophores. Putative sum formulas suggest most of these compounds were not structurally related to each other. We also found that the novel metallophores readily formed complexes with aluminium and were less specific for iron than the ferrioxamines. In our incubations of Trichodesmium colonies, the abundance of ten of the novel metallophores positively correlated with Trichodesmium biomass, but not with bacterial biomass, whilst ferrioxamine siderophores were more strongly associated with bacterial biomass. We identified ferrioxamines and our novel metallophores in filtered surface seawater samples from the Gulf of Aqaba. However, our novel metallophores were only observed in the surface seawater sample collected at the time of highest Trichodesmium abundance, while ferrioxamines were observed even when Trichodesmium was not present. We hypothesize that the novel metallophores were specifically associated with Trichodesmium colonies. Together with the bacterially produced ferrioxamines they likely contribute to a distinctive "ligandosphere" surrounding the Trichodesmium colonies, with potential implications for metal homeostasis within the colony environment.
Assuntos
Desferroxamina/metabolismo , Compostos Férricos/metabolismo , Sideróforos/metabolismo , Trichodesmium/metabolismo , Oceano Índico , Água do Mar/microbiologia , Sideróforos/análise , Trichodesmium/químicaRESUMO
More than 500 siderophores that bind ferric iron have been characterized and grouped by type based on their chemical structure. The chrome azurol S (CAS) assay is a universal colorimetric method that detects siderophores independent of their structure. In this assay, siderophores scavenge iron from an Fe-CAS-hexadecyltrimethylammonium bromide complex, and subsequent release of the CAS dye results in a color change from blue to orange. Solution-based experiments with CAS result in a quantitative measure of siderophore production, while an observable color change on CAS agar plates can be performed for qualitative detection of siderophores. Cross-feeding assays are another useful method to detect and characterize siderophores produced by bacteria. Under iron-limiting conditions, cross-feeding assays test the ability of an indicator strain to grow when supplied with a specific siderophore (from a test strain) to which it has a cognate receptor required for import into the cell. The cross-feeding assay can be tested with a variety of wild-type strains, siderophore biosynthesis mutants, and siderophore receptor mutants.
Assuntos
Hidroxibenzoatos/química , Proteus mirabilis/metabolismo , Sideróforos/análise , Técnicas Bacteriológicas , Calorimetria , Meios de Cultura/química , Sideróforos/químicaRESUMO
This study faces the characterization of the culturable microbiota of the facultative Ni-hyperaccumulator Alyssoides utriculata to obtain a collection of bacterial and fungal strains for potential applications in Ni phytoextraction. Rhizosphere soil samples and adjacent bare soil associated with A. utriculata from serpentine and non-serpentine sites were collected together with plant roots and shoots. Rhizobacteria and fungi were isolated and characterized genotypically and phenotypically. Plants and soils were analyzed for total element concentration using Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Serpentine and non-serpentine sites differ in terms of elements concentration in soil, plant roots and shoots. Ni and Co are significantly higher on serpentine site, while Ca is more abundant in non-serpentine site. Bacteria and fungi were significantly more abundant in rhizosphere than in bare soil and were dominated by genera Arthrobacter, Bacillus and Streptomyces, Penicillium and Mucor. The genus Pseudomonas was only found in rhizospheric serpentine soils (<2% of total serpentine isolates) and with Streptomyces sp. showed highest Ni-tolerance up to 15â¯mM. The same occurred for Trichoderma strain, belonging to the harzianum group (<2% of the total microfungal count) and Penicillium ochrochloron (<10% of the total microfungal count, tolerance up to Ni 20â¯mM). Among serpentine bacterial isolates, 8 strains belonging to 5 genera showed at least one PGPR activity (1-Aminocyclopropane-1-Carboxylic Acid (ACC) deaminase activity, production of indole-3-acetic acid (IAA), siderophores and phosphate solubilizing capacity), especially genera Pantoea, Pseudomonas and Streptomyces. Those microorganisms might thus be promising candidates for employment in bioaugmentation trials.
Assuntos
Níquel/análise , Rizosfera , Poluentes do Solo/análise , Aminoácidos Cíclicos , Bacillus/isolamento & purificação , Bactérias , Brassicaceae/microbiologia , Ácidos Indolacéticos , Raízes de Plantas/química , Pseudomonas , Sideróforos/análise , Solo/química , Microbiologia do SoloRESUMO
Siderophores are chemically diverse small molecules produced by microorganisms for chelation of irons to maintain their survival and govern some important biological functions, especially those cause that infections in hosts. Still, siderophores can offer new insight into a better understanding of the diagnosis and treatments of infectious diseases from the siderophore biosynthesis and regulation perspective. Thus, this review aims to summarize the biomedical value and applicability of siderophores in pathogenic contexts by briefly reviewing mass spectrometry (MS)-based chemical biology and translational applications that involve diagnosis, pathogenesis, and therapeutic discovery for a variety of infectious conditions caused by different pathogens. We highlight the advantages and disadvantages of siderophore discovery and applications in pathogenic contexts. Finally, we propose a panel of new and promising strategy as precision-modification metabolomics method, to rapidly advance the discovery of and translational innovations pertaining to these value compounds in broad biomedical niches. © 2018 Wiley Periodicals, Inc. Mass Spec Rev XX:XX-XX, 2018.
Assuntos
Espectrometria de Massas/métodos , Metabolômica/métodos , Sideróforos/metabolismo , Animais , Infecções Bacterianas/diagnóstico , Infecções Bacterianas/microbiologia , Biomarcadores/análise , Biomarcadores/metabolismo , Vias Biossintéticas , Humanos , Infecções/diagnóstico , Infecções/microbiologia , Micoses/diagnóstico , Micoses/microbiologia , Sideróforos/análise , Pesquisa Translacional Biomédica/métodosRESUMO
The chemical structure of organic molecules profoundly impacts their interactions with metal ions and mineral phases in soils. Understanding the sources and cycling of metal-chelating compounds is therefore essential for predicting the bioavailability and transport of metals throughout terrestrial environments. Here we investigate the molecular speciation of organic molecules that solubilize trace metals in calcareous soils from Eastern Washington. Ultra-high performance Fourier transform ion cyclotron resonance mass spectrometry at 21 Tesla enabled fast and confident detection and identification of metal chelators that are produced by microbes that inhabit these soils based on screening for features that match diagnostic metal isotope patterns. We compared two approaches, one based on direct infusion using the incorporation of a rare isotope to validate true iron-binding features, and another based on separation with liquid chromatography and detection of isotopologues with coherent elution profiles. While the isotopic exchange method requires significantly shorter analysis time, nearly twice as many features were observed with liquid chromatography mass spectrometry (LCMS), mostly due to the reduction in ion suppression where major features limit the sensitivity of minor features. In addition, LCMS enabled the collection of higher quality fragmentation spectra and facilitated feature identification. Siderophores belonging to four major classes were identified, including ferrioxamines, pseudobactins, enterobactins, and arthrobactins. Each of these siderophores likely derives from a unique member of the microbial community, and each possesses different chemical characteristics and uptake pathways, likely contributing to fierce competition for iron within these soils. Our results provide insight into the metabolic pathways by which microbes that co-inhabit calcareous soils compete for this essential micronutrient.
Assuntos
Espectrometria de Massas/métodos , Sideróforos/análise , Microbiologia do Solo , Cromatografia Líquida/métodos , Ciclotrons , Análise de Fourier , Espectrometria de Massas/instrumentaçãoRESUMO
A sobrecarga de ferro é uma condição prejudicial para os pacientes, que apresentam uma diminuição significativa na qualidade de vida. Os fármacos quelantes são moléculas que têm capacidade de uso clínico para atuar como atenuadores da sobrecarga de metais. Neste trabalho apresentamos uma análise de sideróforos do tipo hidroxamato e quinona, com o objetivo de ampliar a gama de terapia de sobrecarga de ferro. Para cada composto foi realizado um ensaio competitivo com a sonda calce- ína para verificar a capacidade de ligação do ferro, e um ensaio antioxidante baseado na supressão da oxidação dependente de ferro da dihidrorrodamina (DHR) sob ascorbato. Foi observado que o hidroxamato cíclico piridoxatina apresentou capacidade de sequestrar ferro de substratos de alta afinidade, tanto em meio tamponado quanto em meio intracelular. Em ambas as situações também se mostrou um antioxidante eficiente. Entretanto, parece ser o mais tóxico do grupo dos hidroxamatos (que ainda continha o hidroxamato linear desferricoprogênio e o aromático desferriastercromo). Outros compostos naturais também foram estudados como possíveis candidatos a fármacos para sobrecarga de ferro. Complexos de ferro foram caracterizados por espectrofotometria para avaliar a estequiometria possível, considerando os sítios de ligação para cada composto. Ensaios de fluorescência revelaram que entre os quatro compostos em estudo (ácido clorogênico, lapachol, hemateína e hematoxilina), o complexo entre ferro e hemateína apresenta maior estabilidade relativa do que outros
Iron overload is a harmful condition for patients, who have a significant decrease in life quality. Chelating drugs are molecules that have the capacity for clinical use to act as attenuators of metal overload. In this work we present an analysis of hydroxamate and quinone-type siderophores, intending to broaden the range of iron overload therapy. For each compound it was conducted a competitive assay with the fluorescent probe calcein to verify the iron binding ability, and an antioxidant assay based on suppression of the iron-dependent oxidation of dihydrorhodamine (DHR) under ascorbate. It was observed that cyclic hydroxamate pyridoxatin displayed good ability to scavenge iron from high affinity substrates both in buffer and in intracellular medium. It was also an efficient antioxidant in both setups. However, pyridoxatin seems to be the most toxic from the hydroxamate group (composed also by the linear desferricoprogen and the aromatic desferriasterchrome). Other natural compounds have also been studied as possible candidates for iron-overload drug therapy. Iron complexes were characterized by spectrophotometry to assess the possible stoichiometry considering the binding sites for each compound. Fluorescence assays revealed that among the four compounds in study (chlorogenic acid, lapachol, hematein and hematoxylin), the complex between iron and hematein has higher relative stability than others
Assuntos
Sideróforos/análise , Sobrecarga de Ferro/terapia , Fluorescência , Espectrofotometria/instrumentação , Terapia por Quelação , Desferroxamina/classificação , Ferro/efeitos adversos , AntioxidantesRESUMO
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.