Using Repeated Lysis Steps Fractionates Between Heterotrophic and Cyanobacterial DNA Extracted from Xenic Cyanobacterial Cultures.

Extracting DNA from cyanobacteria can be a challenge because of their diverse morphologies, challenging cellular structure, and the heterotrophic microbiome often present within cyanobacterial cultures. As such, even when our DNA yields are sufficient for sequencing, the percentage of reads coming from the cyanobacterial host can be low, leading to incomplete genomes spread across several scaffolds. In this research, we optimized a DNA isolation protocol using three iterative cell lysis steps to enrich the portion of DNA isolated coming from the cyanobacterial host rather than the heterotrophic microbiome.

LCMS-Metabolomic Profiling and Genome Mining of DSM 21246 Revealed Lipophilic Delftibactin Metallophores.

Bacteria have evolved various strategies to combat heavy metal stress, including the secretion of small molecules, known as metallophores. These molecules hold a potential role in the mitigation of toxic metal contamination from the environment (bioremediation). Herein, we employed combined comparative metabolomic and genomic analyses to study the metallophores excreted by DSM 21246.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from Marine Cyanobacterium JHB Discovered via Coculture with .

The tropical marine cyanobacterium JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies on this strain led to the discovery of several novel compounds such as hectochlorins and jamaicamides. However, bioinformatic analyses of its genome indicate the presence of numerous cryptic biosynthetic gene clusters that have yet to be characterized.

Comparative metabolomic profiling of B-4383 revealed production of cupriachelin siderophores, one with activity against .

H16 is known to be a rich source of linear lipopeptide siderophores when grown under iron-depleted conditions; prior literature termed these compounds cupriachelins. These small molecules bear -hydroxyaspartate moieties that contribute to a photoreduction of iron when bound as ferric cupriachelin. Here, we present structural assignment of cupriachelins from B-4383 grown under iron limitation.

Structure and Biosynthesis of Hectoramide B, a Linear Depsipeptide from the Marine Cyanobacterium JHB Discovered via Co-culture with .

The tropical marine cyanobacterium JHB is a prolific source of secondary metabolites with potential biomedical utility. Previous studies of this strain led to the discovery of several novel compounds such as the hectochlorins and jamaicamides; however, bioinformatic analyses of its genome suggested that there were many more cryptic biosynthetic gene clusters yet to be characterized. To potentially stimulate the production of novel compounds from this strain, it was co-cultured with .

Luquilloamides, Cytotoxic Lipopeptides from a Puerto Rican Collection of the Filamentous Marine Cyanobacterium sp.

Luquilloamides A-G (-) were isolated from a small environmental collection of a marine cyanobacterium found growing on eelgrass ( sp.) near Luquillo, Puerto Rico. Structure elucidation of the luquilloamides was accomplished via detailed NMR and MS analyses, and absolute configurations were determined using a combination of advanced Mosher's method, -based configuration analysis, semisynthetic fragment analysis derived from ozonolysis, methylation, Baeyer-Villiger oxidation, Mosher's esterification, specific rotations, and ECD data.

Design of Gallinamide A Analogs as Potent Inhibitors of the Cysteine Proteases Human Cathepsin L and Cruzain.

Gallinamide A, originally isolated with a modest antimalarial activity, was subsequently reisolated and characterized as a potent, selective, and irreversible inhibitor of the human cysteine protease cathepsin L. Molecular docking identified potential modifications to improve binding, which were synthesized as a suite of analogs. Resultingly, this current study produced the most potent gallinamide analog yet tested against cathepsin L (, = 0.

Reactivity of an Unusual Amidase May Explain Colibactin's DNA Cross-Linking Activity.

Certain commensal and pathogenic bacteria produce colibactin, a small-molecule genotoxin that causes interstrand cross-links in host cell DNA. Although colibactin alkylates DNA, the molecular basis for cross-link formation is unclear. Here, we report that the colibactin biosynthetic enzyme ClbL is an amide bond-forming enzyme that links aminoketone and β-keto thioester substrates in vitro and in vivo.

The human gut bacterial genotoxin colibactin alkylates DNA.

Certain strains residing in the human gut produce colibactin, a small-molecule genotoxin implicated in colorectal cancer pathogenesis. However, colibactin's chemical structure and the molecular mechanism underlying its genotoxic effects have remained unknown for more than a decade. Here we combine an untargeted DNA adductomics approach with chemical synthesis to identify and characterize a covalent DNA modification from human cell lines treated with colibactin-producing Our data establish that colibactin alkylates DNA with an unusual electrophilic cyclopropane.

Marine Natural Product Honaucin A Attenuates Inflammation by Activating the Nrf2-ARE Pathway.

The cyanobacterial marine natural product honaucin A inhibits mammalian innate inflammation in vitro and in vivo. To decipher its mechanism of action, RNA sequencing was used to evaluate differences in gene expression of cultured macrophages following honaucin A treatment. This analysis led to the hypothesis that honaucin A exerts its anti-inflammatory activity through activation of the cytoprotective nuclear erythroid 2-related factor 2 (Nrf2)-antioxidant response element/electrophile response element (ARE/EpRE) signaling pathway.

Integrating Molecular Networking and Biological Assays To Target the Isolation of a Cytotoxic Cyclic Octapeptide, Samoamide A, from an American Samoan Marine Cyanobacterium.

Integrating LC-MS/MS molecular networking and bioassay-guided fractionation enabled the targeted isolation of a new and bioactive cyclic octapeptide, samoamide A (1), from a sample of cf. Symploca sp. collected in American Samoa.

Sharing and community curation of mass spectrometry data with Global Natural Products Social Molecular Networking.

The potential of the diverse chemistries present in natural products (NP) for biotechnology and medicine remains untapped because NP databases are not searchable with raw data and the NP community has no way to share data other than in published papers. Although mass spectrometry (MS) techniques are well-suited to high-throughput characterization of NP, there is a pressing need for an infrastructure to enable sharing and curation of data. We present Global Natural Products Social Molecular Networking (GNPS; http://gnps.

Synthesis of Highly Substituted Quinolines via a Tandem Ynamide Benzannulation/Iodocyclization Strategy.

A two-stage "tandem strategy" for the regiocontrolled synthesis of very highly substituted quinolines is described. Benzannulation based on the reaction of cyclobutenones or diazo ketones with N-propargyl-substituted ynamides proceeds via a cascade of several pericyclic reactions to generate multiply substituted aniline derivatives. In the second stage of the tandem strategy, triflate derivatives of the phenolic benzannulation products undergo Larock cyclization upon exposure to iodine to form products that are further elaborated by methods such as palladium-catalyzed coupling to generate quinolines that can be substituted at every position of the bicyclic system.

Expanding the Described Metabolome of the Marine Cyanobacterium Moorea producens JHB through Orthogonal Natural Products Workflows.

Moorea producens JHB, a Jamaican strain of tropical filamentous marine cyanobacteria, has been extensively studied by traditional natural products techniques. These previous bioassay and structure guided isolations led to the discovery of two exciting classes of natural products, hectochlorin (1) and jamaicamides A (2) and B (3). In the current study, mass spectrometry-based 'molecular networking' was used to visualize the metabolome of Moorea producens JHB, and both guided and enhanced the isolation workflow, revealing additional metabolites in these compound classes.

Lipopeptides from the tropical marine cyanobacterium Symploca sp.

A collection of the tropical marine cyanobacterium Symploca sp., collected near Kimbe Bay, Papua New Guinea, previously yielded several new metabolites including kimbeamides A-C, kimbelactone A, and tasihalide C. Investigations into a more polar cytotoxic fraction yielded three new lipopeptides, tasiamides C-E (1-3).

MS/MS-based networking and peptidogenomics guided genome mining revealed the stenothricin gene cluster in Streptomyces roseosporus.

Most (75%) of the anti-infectives that save countless lives and enormously improve quality of life originate from microbes found in nature. Herein, we described a global visualization of the detectable molecules produced from a single microorganism, which we define as the 'molecular network' of that organism, followed by studies to characterize the cellular effects of antibacterial molecules. We demonstrate that Streptomyces roseosporus produces at least four non-ribosomal peptide synthetase-derived molecular families and their gene subnetworks (daptomycin, arylomycin, napsamycin and stenothricin) were identified with different modes of action.

Molecular networking as a dereplication strategy.

A major goal in natural product discovery programs is to rapidly dereplicate known entities from complex biological extracts. We demonstrate here that molecular networking, an approach that organizes MS/MS data based on chemical similarity, is a powerful complement to traditional dereplication strategies. Successful dereplication with molecular networks requires MS/MS spectra of the natural product mixture along with MS/MS spectra of known standards, synthetic compounds, or well-characterized organisms, preferably organized into robust databases.

Viequeamide A, a cytotoxic member of the kulolide superfamily of cyclic depsipeptides from a marine button cyanobacterium.

The viequeamides, a family of 2,2-dimethyl-3-hydroxy-7-octynoic acid (Dhoya)-containing cyclic depsipeptides, were isolated from a shallow subtidal collection of a "button" cyanobacterium (Rivularia sp.) from near the island of Vieques, Puerto Rico. Planar structures of the two major compounds, viequeamide A (1) and viequeamide B (2), were elucidated by 2D-NMR spectroscopy and mass spectrometry, whereas absolute configurations were determined by traditional hydrolysis, derivative formation, and chromatography in comparison with standards.