RESUMO
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.
Assuntos
Elementos da Série dos Lantanídeos , Methylobacterium extorquens , Elementos da Série dos Lantanídeos/metabolismo , Elementos da Série dos Lantanídeos/química , Methylobacterium extorquens/metabolismo , Methylobacterium extorquens/genéticaRESUMO
Microorganisms produce small bioactive compounds as part of their secondary or specialised metabolism. Often, such metabolites have antimicrobial, anticancer, antifungal, antiviral or other bio-activities and thus play an important role for applications in medicine and agriculture. In the past decade, genome mining has become a widely-used method to explore, access, and analyse the available biodiversity of these compounds. Since 2011, the 'antibiotics and secondary metabolite analysis shell-antiSMASH' (https://antismash.secondarymetabolites.org/) has supported researchers in their microbial genome mining tasks, both as a free to use web server and as a standalone tool under an OSI-approved open source licence. It is currently the most widely used tool for detecting and characterising biosynthetic gene clusters (BGCs) in archaea, bacteria, and fungi. Here, we present the updated version 7 of antiSMASH. antiSMASH 7 increases the number of supported cluster types from 71 to 81, as well as containing improvements in the areas of chemical structure prediction, enzymatic assembly-line visualisation and gene cluster regulation.
Assuntos
Computadores , Software , Bactérias/genética , Bactérias/metabolismo , Archaea/genética , Genoma Microbiano , Família Multigênica , Metabolismo Secundário/genéticaRESUMO
With an ever-increasing amount of (meta)genomic data being deposited in sequence databases, (meta)genome mining for natural product biosynthetic pathways occupies a critical role in the discovery of novel pharmaceutical drugs, crop protection agents and biomaterials. The genes that encode these pathways are often organised into biosynthetic gene clusters (BGCs). In 2015, we defined the Minimum Information about a Biosynthetic Gene cluster (MIBiG): a standardised data format that describes the minimally required information to uniquely characterise a BGC. We simultaneously constructed an accompanying online database of BGCs, which has since been widely used by the community as a reference dataset for BGCs and was expanded to 2021 entries in 2019 (MIBiG 2.0). Here, we describe MIBiG 3.0, a database update comprising large-scale validation and re-annotation of existing entries and 661 new entries. Particular attention was paid to the annotation of compound structures and biological activities, as well as protein domain selectivities. Together, these new features keep the database up-to-date, and will provide new opportunities for the scientific community to use its freely available data, e.g. for the training of new machine learning models to predict sequence-structure-function relationships for diverse natural products. MIBiG 3.0 is accessible online at https://mibig.secondarymetabolites.org/.
Assuntos
Genoma , Genômica , Família Multigênica , Vias Biossintéticas/genéticaRESUMO
The Gram-negative bacterium Yersinia ruckeri is the causative agent for enteric red mouth disease in salmonids. The genome of Y. ruckeri YRB contains a biosynthetic gene cluster encoding the biosynthesis of catechol siderophores that are diastereomeric with the known vanchrobactin class of siderophores, (DHBDArgLSer)(1-3). Ruckerbactin (1), produced by Y. ruckeri YRB, was found to be the linear tris-l-serine ester composed of l-arginine and 2,3-dihydroxybenzoic acid, (DHBLArgLSer)3. The biscatechol, (DHBLArgLSer)2 (2), and monocatechol, DHBLArgLSer (3), compounds were also isolated and characterized. The macrolactone of ruckerbactin was not detected. The presence of LArg in ruckerbactin makes it the diastereomer of trivanchrobactin with DArg. The electronic circular dichroism spectra of Fe(III)-ruckerbactin and Fe(III)-trivanchrobactin reveal the opposite enantiomeric configurations at the Fe(III) sites. Fe(III)-ruckerbactin adopts the Δ configuration, and Fe(III)-trivanchrobactin adopts the Λ configuration. Y. ruckeri YRB was also found to produce the antimicrobial agent holomycin (4).
Assuntos
Peptídeos , Vibrio , Yersinia ruckeri , Dicroísmo Circular , Peptídeos/metabolismo , Sideróforos , Análise Espectral/métodos , Estereoisomerismo , Vibrio/metabolismo , Yersinia ruckeri/metabolismoRESUMO
Genome mining of biosynthetic pathways streamlines discovery of secondary metabolites but can leave ambiguities in the predicted structures, which must be rectified experimentally. Through coupling the reactivity predicted by biosynthetic gene clusters with verified structures, the origin of the ß-hydroxyaspartic acid diastereomers in siderophores is reported herein. Two functional subtypes of nonheme Fe(II)/α-ketoglutarate-dependent aspartyl ß-hydroxylases are identified in siderophore biosynthetic gene clusters, which differ in genomic organization-existing either as fused domains (IßHAsp) at the carboxyl terminus of a nonribosomal peptide synthetase (NRPS) or as stand-alone enzymes (TßHAsp)-and each directs opposite stereoselectivity of Asp ß-hydroxylation. The predictive power of this subtype delineation is confirmed by the stereochemical characterization of ß-OHAsp residues in pyoverdine GB-1, delftibactin, histicorrugatin, and cupriachelin. The l-threo (2S, 3S) ß-OHAsp residues of alterobactin arise from hydroxylation by the ß-hydroxylase domain integrated into NRPS AltH, while l-erythro (2S, 3R) ß-OHAsp in delftibactin arises from the stand-alone ß-hydroxylase DelD. Cupriachelin contains both l-threo and l-erythro ß-OHAsp, consistent with the presence of both types of ß-hydroxylases in the biosynthetic gene cluster. A third subtype of nonheme Fe(II)/α-ketoglutarate-dependent enzymes (IßHHis) hydroxylates histidyl residues with l-threo stereospecificity. A previously undescribed, noncanonical member of the NRPS condensation domain superfamily is identified, named the interface domain, which is proposed to position the ß-hydroxylase and the NRPS-bound amino acid prior to hydroxylation. Through mapping characterized ß-OHAsp diastereomers to the phylogenetic tree of siderophore ß-hydroxylases, methods to predict ß-OHAsp stereochemistry in silico are realized.
Assuntos
Bactérias/enzimologia , Oxigenases de Função Mista/genética , Sideróforos/genética , Sideróforos/metabolismo , Ácido Aspártico/química , Vias Biossintéticas , Quelantes/farmacologia , Genoma Bacteriano , Genômica , Ferro/metabolismo , Funções Verossimilhança , Oxigenases de Função Mista/metabolismo , Família Multigênica , Peptídeo Sintases/química , Peptídeo Sintases/genética , Filogenia , Estereoisomerismo , Especificidade por SubstratoRESUMO
DNA methylation is a heritable epigenetic modification that plays an essential role in mammalian development. Genomic methylation patterns are dynamically maintained, with DNA methyltransferases mediating inheritance of methyl marks onto nascent DNA over cycles of replication. A recently developed experimental technique employing immunoprecipitation of bromodeoxyuridine labeled nascent DNA followed by bisulfite sequencing (Repli-BS) measures post-replication temporal evolution of cytosine methylation, thus enabling genome-wide monitoring of methylation maintenance. In this work, we combine statistical analysis and stochastic mathematical modeling to analyze Repli-BS data from human embryonic stem cells. We estimate site-specific kinetic rate constants for the restoration of methyl marks on >10 million uniquely mapped cytosines within the CpG (cytosine-phosphate-guanine) dinucleotide context across the genome using Maximum Likelihood Estimation. We find that post-replication remethylation rate constants span approximately two orders of magnitude, with half-lives of per-site recovery of steady-state methylation levels ranging from shorter than ten minutes to five hours and longer. Furthermore, we find that kinetic constants of maintenance methylation are correlated among neighboring CpG sites. Stochastic mathematical modeling provides insight to the biological mechanisms underlying the inference results, suggesting that enzyme processivity and/or collaboration can produce the observed kinetic correlations. Our combined statistical/mathematical modeling approach expands the utility of genomic datasets and disentangles heterogeneity in methylation patterns arising from replication-associated temporal dynamics versus stable cell-to-cell differences.
Assuntos
Biologia Computacional/métodos , Metilação de DNA/fisiologia , Animais , Bromodesoxiuridina/química , Ilhas de CpG , Citosina/metabolismo , DNA/metabolismo , Metilases de Modificação do DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética/genética , Epigênese Genética/fisiologia , Epigenômica/métodos , Genoma , Genômica , Humanos , Cinética , Modelos Estatísticos , Modelos Teóricos , Processos EstocásticosRESUMO
Vibrio campbellii BAA-1116 (formerly Vibrio harveyi) is a model organism for quorum sensing study and produces the siderophores anguibactin and amphi-enterobactin. This study examined the mechanisms and specificity of siderophore uptake in V. campbellii and V. harveyi, and surveyed the diversity of siderophore production in V. campbellii and V. harveyi strains. The amphi-enterobactin gene cluster of BAA-1116 harbors a gene, named fapA, that is a homologue of genes encoding Fe(III)-siderophore-specific outer membrane receptors. Another strain, V. campbellii HY01, a strain pathogenic to shrimp, also carries this cluster including fapA. Our siderophore bioassay results using HY01-derived indicator strains show that the FapA protein localized in the outer membrane fraction of V. campbellii HY01 is essential for the uptake of Fe(III)-amphi-enterobactin as well as exogenous siderophores, including enterobactin from E. coli, but not vanchrobactin from V. anguillarum RV22 while Fe(III)-amphi-enterobactin can be utilized by V. anguillarum. Electrospray ionization mass spectrometry as well as bioassay revealed that various V. campbellii and V. harveyi strains produce a suite of amphi-enterobactins with various fatty acid appendages, including several novel amphi-enterobactins, and these amphi-enterobactins can be taken up by V. campbellii HY01 via FapA, indicating that amphi-enterobactin production is a common phenotype among V. campbellii and V. harveyi, whereas our previous work, confirmed herein, showed that anguibactin is only produced by V. campbellii strains. These results along with the additional finding that a 2,3-dihydroxybenzoic acid biosynthesis gene, aebA, located in the amphi-enterobactin gene cluster, is essential for both anguibactin and amphi-enterobactin biosynthesis, suggest the possibility that amphi-enterobactin is a native siderophore of V. campbellii and V. harveyi, while the anguibactin system has been acquired by V. campbellii during evolution.
Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Enterobactina/metabolismo , Compostos Férricos/metabolismo , Vibrio/química , Proteínas da Membrana Bacteriana Externa/química , Enterobactina/biossíntese , Enterobactina/química , Compostos Férricos/química , Conformação Molecular , Vibrio/metabolismoRESUMO
In the original publication, third author's name was incorrectly published as Aneta L. Jelowicki.
RESUMO
The vast majority of bacteria require iron to grow. A significant iron acquisition strategy is the production of siderophores, which are secondary microbial metabolites synthesized to sequester iron(III). Siderophore structures encompass a variety of forms, of which highly modified peptidic siderophores are of interest herein. State-of-the-art genome mining tools, such as antiSMASH (antibiotics & Secondary Metabolite Analysis SHell), hold the potential to predict and discover new peptidic siderophores, including a combinatoric suite of triscatechol siderophores framed on a triserine-ester backbone of the general class, (DHB-l/d CAA-l Ser)3 (CAA, cationic amino acid). Siderophores with l/d Arg, l/d Lys and l Orn, but not d Orn, were predicted in bacterial genomes. Fortuitously the d Orn siderophore was identified, yet its lack of prediction highlights the limitation of current genome mining tools. The full combinatoric suite of these siderophores, which form chiral iron(III) complexes, reveals stereospecific coordination chemistry encoded in microbial genomes. The chirality embedded in this suite of Fe(III)-siderophores raises the question of whether the relevant siderophore-mediated iron acquisition pathways are stereospecific and selective for ferric siderophore complexes of a defined configuration.
Assuntos
Compostos Férricos , Sideróforos , Sideróforos/genética , Compostos Férricos/metabolismo , Ferro/metabolismo , Bactérias/metabolismo , Genoma Bacteriano , Peptídeos/genética , Peptídeos/metabolismoRESUMO
Ingestion of the cycad toxins ß-methylamino-L-alanine (BMAA) and azoxyglycosides is harmful to diverse organisms. However, some insects are specialized to feed on toxin-rich cycads with apparent immunity. Some cycad-feeding insects possess a common set of gut bacteria, which might play a role in detoxifying cycad toxins. Here, we investigated the composition of gut microbiota from a worldwide sample of cycadivorous insects and characterized the biosynthetic potential of selected bacteria. Cycadivorous insects shared a core gut microbiome consisting of six bacterial taxa, mainly belonging to the Proteobacteria, which we were able to isolate. To further investigate selected taxa from diverging lineages, we performed shotgun metagenomic sequencing of co-cultured bacterial sub-communities. We characterized the biosynthetic potential of four bacteria from Serratia, Pantoea, and two different Stenotrophomonas lineages, and discovered a suite of biosynthetic gene clusters notably rich in siderophores. Siderophore semi-untargeted metabolomics revealed a broad range of chemically related yet diverse iron-chelating metabolites, including desferrioxamine B, suggesting the occurrence of an unprecedented desferrioxamine-like biosynthetic pathway that remains to be identified. These results provide a foundation for future investigations into how cycadivorous insects tolerate diets rich in azoxyglycosides, BMAA, and other cycad toxins, including a possible role for bacterial siderophores.
RESUMO
Many bacteria use small-molecule chelators called metallophores to acquire trace metals from their environment. These molecules play a central role in interactions between bacteria, plants, and animals. Hence, knowing their full diversity is key to combatting infectious diseases as well as harnessing beneficial microbial communities. Metallophore discovery has been streamlined by advances in genome mining, where genomes are scanned for genes involved in metallophore biosynthesis. This review highlights recent trends and advances in predicting the presence and structure of metallophores based solely on genomic information. Recent work suggests new families of metallophores remain hidden from current homology-based approaches. Their discovery will require new genome mining approaches that move beyond biosynthesis to consider metallophore transporters, regulation, and evolution.
Assuntos
Bactérias , Bactérias/genéticaRESUMO
Resolving the spatial distribution of RNA and protein in tissues at subcellular resolution is a challenge in the field of spatial biology. We describe spatial molecular imaging, a system that measures RNAs and proteins in intact biological samples at subcellular resolution by performing multiple cycles of nucleic acid hybridization of fluorescent molecular barcodes. We demonstrate that spatial molecular imaging has high sensitivity (one or two copies per cell) and very low error rate (0.0092 false calls per cell) and background (~0.04 counts per cell). The imaging system generates three-dimensional, super-resolution localization of analytes at ~2 million cells per sample. Cell segmentation is morphology based using antibodies, compatible with formalin-fixed, paraffin-embedded samples. We measured multiomic data (980 RNAs and 108 proteins) at subcellular resolution in formalin-fixed, paraffin-embedded tissues (nonsmall cell lung and breast cancer) and identified >18 distinct cell types, ten unique tumor microenvironments and 100 pairwise ligand-receptor interactions. Data on >800,000 single cells and ~260 million transcripts can be accessed at http://nanostring.com/CosMx-dataset .
Assuntos
Proteínas , RNA , Humanos , Inclusão em Parafina , RNA/genética , Imagem Molecular , FormaldeídoRESUMO
Ferric complexes of triscatechol siderophores may assume one of two enantiomeric configurations at the iron site. Chirality is known to be important in the iron uptake process, however an understanding of the molecular features directing stereospecific coordination remains ambiguous. Synthesis of the full suite of (DHBL/DLysL/DSer)3 macrolactone diastereomers, which includes the siderophore cyclic trichrysobactin (CTC), enables the effects that the chirality of Lys and Ser residues exert on the configuration of the Fe(iii) complex to be defined. Computationally optimized geometries indicate that the Λ/Δ configurational preferences are set by steric interactions between the Lys sidechains and the peptide backbone. The ability of each (DHBL/DLysL/DSer)3 diastereomer to form a stable Fe(iii) complex prompted a genomic search for biosynthetic gene clusters (BGCs) encoding the synthesis of these diastereomers in microbes. The genome of the plant pathogen Dickeya chrysanthemi EC16 was sequenced and the genes responsible for the biosynthesis of CTC were identified. A related but distinct BGC was identified in the genome of the opportunistic pathogen Yersinia frederiksenii ATCC 33641; isolation of the siderophore from Y. frederiksenii ATCC 33641, named frederiksenibactin (FSB), revealed the triscatechol oligoester, linear-(DHBLLysLSer)3. Circular dichroism (CD) spectroscopy establishes that Fe(iii)-CTC and Fe(iii)-FSB are formed in opposite enantiomeric configuration, consistent with the results of the ferric complexes of the cyclic (DHBL/DLysL/DSer)3 diastereomers.
RESUMO
Genome mining for VibH homologs reveals several species of Acinetobacter with a gene cluster that putatively encodes the biosynthesis of catechol siderophores with an amine core. A. bouvetii DSM 14964 produces three novel biscatechol siderophores: propanochelin (1), butanochelin (2), and pentanochelin (3). This strain has a relaxed specificity for the amine substrate, allowing for the biosynthesis of a variety of non-natural siderophore analogs by precursor directed biosynthesis. Of potential synthetic utility, A. bouvetii DSM 14964 condenses 2,3-dihydroxybenzoic acid (2,3-DHB) to allylamine and propargylamine, producing catecholic compounds which bind iron(iii) and may be further modified via thiol-ene or azide-alkyne click chemistry.
Assuntos
Acinetobacter/química , Catecóis/metabolismo , Acinetobacter/metabolismo , Catecóis/química , Estrutura Molecular , EstereoisomerismoRESUMO
Macrophages perform critical functions for homeostasis and immune defense in tissues throughout the body. These innate immune cells are capable of recognizing and clearing dead cells and pathogens, and orchestrating inflammatory and healing processes that occur in response to injury. In addition, macrophages are involved in the progression of many inflammatory diseases including cardiovascular disease, fibrosis, and cancer. Although it has long been known that macrophages respond dynamically to biochemical signals in their microenvironment, the role of biophysical cues has only recently emerged. Furthermore, many diseases that involve macrophages are also characterized by changes to the tissue biophysical environment. This review will discuss current knowledge about the effects of biophysical cues including matrix stiffness, material topography, and applied mechanical forces, on macrophage behavior. We will also describe the role of molecules that are known to be important for mechanotransduction, including adhesion molecules, ion channels, as well as nuclear mediators such as transcription factors, scaffolding proteins, and epigenetic regulators. Together, this review will illustrate a developing role of biophysical cues in macrophage biology, and also speculate upon molecular targets that may potentially be exploited therapeutically to treat disease.
Assuntos
Suscetibilidade a Doenças , Fenômenos do Sistema Imunitário , Imunomodulação , Ativação de Macrófagos/imunologia , Macrófagos/imunologia , Macrófagos/metabolismo , Animais , Adesão Celular , Sinais (Psicologia) , Epigênese Genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica , Humanos , Canais Iônicos/metabolismo , Lipopolissacarídeos/imunologia , Macrófagos/citologia , Mecanotransdução Celular , Transdução de SinaisRESUMO
Bacteria often produce siderophores to facilitate iron uptake. One of the most studied siderophores is enterobactin, the macrolactone trimer of 2,3-dihydroxybenzoyl-l-serine, produced by E. coli and many other enteric bacteria. Other siderophores are variants of enterobactin, with structural modifications including expansion of the tri-serine core to a tetra-serine macrolactone, substitution of l-serine with l-threonine, insertion of amino acids (i.e., Gly, l-Ala, d-Lys, d- and l-Arg, l-Orn), catechol glucosylation, and linearization of the tri-serine macrolactone core. In this review we summarize the current understanding of the biosyntheses of these enterobactin variants, placing them in contrast with the well-established biosynthesis of enterobactin.