Outlining the Hidden Curriculum: Perspectives on Successfully Navigating Scientific Conferences.

Scientific conferences and meetings are valuable opportunities for researchers to network, communicate, and develop knowledge. For early career scientists, conferences can also be intimidating, confusing, and overwhelming, especially without having adequate preparation or experience. In this Perspective, we provide advice based on previous experiences navigating scientific meetings and conferences. These guidelines outline parts of the hidden curriculum around preparing for and attending meetings, navigating conference sessions, networking with other scientists, and participating in social activities while upholding a recommended code of conduct.

The Discovery of Acremochlorins O-R from an Acremonium sp. through Integrated Genomic and Molecular Networking.

The fermentation of a soil-derived fungus Acremonium sp. led to the isolation of thirteen ascochlorin congeners through integrated genomic and Global Natural Product Social (GNPS) molecular networking. Among the isolated compounds, we identified two unusual bicyclic types, acremochlorins O (1) and P (2), as well as two linear types, acremochlorin Q (3) and R (4). Compounds 1 and 2 contain an unusual benzopyran moiety and are diastereoisomers of each other, the first reported for the ascochlorins. Additionally, we elucidated the structure of 5, a 4-chloro-5-methylbenzene-1,3-diol with a linear farnesyl side chain, and confirmed the presence of eight known ascochlorin analogs (6-13). The structures were determined by the detailed interpretation of 1D and 2D NMR spectroscopy, MS, and ECD calculations. Compounds 3 and 9 showed potent antibacterial activity against Staphylococcus aureus and Bacillus cereus, with MIC values ranging from 2 to 16 µg/mL.

Bacterial diversity and chemical ecology of natural product-producing bacteria from Great Salt Lake sediment.

Great Salt Lake (GSL), located northwest of Salt Lake City, UT, is the largest terminal lake in the USA. While the average salinity of seawater is ~3.3%, the salinity in GSL ranges between 5% and 28%. In addition to being a hypersaline environment, GSL also contains toxic concentrations of heavy metals, such as arsenic, mercury, and lead. The extreme environment of GSL makes it an intriguing subject of study, both for its unique microbiome and its potential to harbor novel natural product-producing bacteria, which could be used as resources for the discovery of biologically active compounds. Though work has been done to survey and catalog bacteria found in GSL, the Lake's microbiome is largely unexplored, and little to no work has been done to characterize the natural product potential of GSL microbes. Here, we investigate the bacterial diversity of two important regions within GSL, describe the first genomic characterization of Actinomycetota isolated from GSL sediment, including the identification of two new Actinomycetota species, and provide the first survey of the natural product potential of GSL bacteria.

Resistance Gene Association and Inference Network (ReGAIN): A Bioinformatics Pipeline for Assessing Probabilistic Co-Occurrence Between Resistance Genes in Bacterial Pathogens.

The rampant rise of multidrug resistant (MDR) bacterial pathogens poses a severe health threat, necessitating innovative tools to unravel the complex genetic underpinnings of antimicrobial resistance. Despite significant strides in developing genomic tools for detecting resistance genes, a gap remains in analyzing organism-specific patterns of resistance gene co-occurrence. Addressing this deficiency, we developed the Resistance Gene Association and Inference Network (ReGAIN), a novel web-based and command line genomic platform that uses Bayesian network structure learning to identify and map resistance gene networks in bacterial pathogens. ReGAIN not only detects resistance genes using well-established methods, but also elucidates their complex interplay, critical for understanding MDR phenotypes. Focusing on ESKAPE pathogens, ReGAIN yielded a queryable database for investigating resistance gene co-occurrence, enriching resistome analyses, and providing new insights into the dynamics of antimicrobial resistance. Furthermore, the versatility of ReGAIN extends beyond antibiotic resistance genes to include assessment of co-occurrence patterns among heavy metal resistance and virulence determinants, providing a comprehensive overview of key gene relationships impacting both disease progression and treatment outcomes.

Structural, biochemical and bioinformatic analyses of nonribosomal peptide synthetase adenylation domains.

Covering: 1997 to July 2023The adenylation reaction has been a subject of scientific intrigue since it was first recognized as essential to many biological processes, including the homeostasis and pathogenicity of some bacteria and the activation of amino acids for protein synthesis in mammals. Several foundational studies on adenylation (A) domains have facilitated an improved understanding of their molecular structures and biochemical properties, in particular work on nonribosomal peptide synthetases (NRPSs). In NRPS pathways, A domains activate their respective acyl substrates for incorporation into a growing peptidyl chain, and many nonribosomal peptides are bioactive. From a natural product drug discovery perspective, improving existing bioinformatics platforms to predict unique NRPS products more accurately from genomic data is desirable. Here, we summarize characterization efforts of A domains primarily from NRPS pathways from July 1997 up to July 2023, covering protein structure elucidation, in vitro assay development, and in silico tools for improved predictions.

Conformational Control in Diterpene Synthase TlnA: Elucidating the Mechanism of 5-8-6 Skeleton Formation through Ring Expansion.

TlnA produces a distinct cyclohexane-fused 5-8-6 ring system, different from the prevalent 5-8-5 scaffold synthesized by well-established enzymes. This study identifies two conformations of a carbocation intermediate, revealing how the enzyme environment prohibits one conformation due to steric hindrance, thereby directing the formation of the 5-8-6 system over the 5-8-5 scaffold. This investigation enhances our understanding of diterpene biosynthesis and the impact of enzyme environments on chemical reactions, providing valuable insights into the formation of complex cyclic structures.

Bacterial Diversity and Chemical Ecology of Natural Product-Producing Bacteria from Great Salt Lake Sediment.

Great Salt Lake (GSL), located northwest of Salt Lake City, UT, is the largest terminal lake in the United States. While the average salinity of seawater is ~3.3%, the salinity in GSL ranges between 5-28%. In addition to being a hypersaline environment, GSL also contains toxic concentrations of heavy metals, such as arsenic, mercury, and lead. The extreme environment of GSL makes it an intriguing subject of study, both for its unique microbiome and its potential to harbor novel natural product-producing bacteria, which could be used as resources for the discovery of biologically active compounds. Though work has been done to survey and catalogue bacteria found in GSL, the Lake's microbiome is largely unexplored, and little-to-no work has been done to characterize the natural product potential of GSL microbes. Here, we investigate the bacterial diversity of two important regions within GSL, describe the first genomic characterization of Actinomycetota isolated from GSL sediment, including the identification of a new Saccharomonospora species, and provide the first survey of the natural product potential of GSL bacteria.

Biosynthesis of the fungal nonribosomal peptide penilumamide A and biochemical characterization of a pterin-specific adenylation domain.

We report the characterization of the penilumamide biosynthetic cluster from Aspergillus flavipes CNL-338. In vitro reconstitution experiments demonstrated that three nonribosomal peptide synthetases are required for constructing the tripeptide and studies with dissected adenylation domains allowed for the first biochemical characterization of a domain that selects a pterin-derived building block.

Expanding the Utility of Bioinformatic Data for the Full Stereostructural Assignments of Marinolides A and B, 24- and 26-Membered Macrolactones Produced by a Chemically Exceptional Marine-Derived Bacterium.

Marinolides A and B, two new 24- and 26-membered bacterial macrolactones, were isolated from the marine-derived actinobacterium AJS-327 and their stereostructures initially assigned by bioinformatic data analysis. Macrolactones typically possess complex stereochemistry, the assignments of which have been one of the most difficult undertakings in natural products chemistry, and in most cases, the use of X-ray diffraction methods and total synthesis have been the major methods of assigning their absolute configurations. More recently, however, it has become apparent that the integration of bioinformatic data is growing in utility to assign absolute configurations. Genome mining and bioinformatic analysis identified the 97 kb mld biosynthetic cluster harboring seven type I polyketide synthases. A detailed bioinformatic investigation of the ketoreductase and enoylreductase domains within the multimodular polyketide synthases, coupled with NMR and X-ray diffraction data, allowed for the absolute configurations of marinolides A and B to be determined. While using bioinformatics to assign the relative and absolute configurations of natural products has high potential, this method must be coupled with full NMR-based analysis to both confirm bioinformatic assignments as well as any additional modifications that occur during biosynthesis.

DNA Authentication and Chemical Analysis of Psilocybe Mushrooms Reveal Widespread Misdeterminations in Fungaria and Inconsistencies in Metabolites.

The mushroom genus Psilocybe is best known as the core group of psychoactive mushrooms, yet basic information on their diversity, taxonomy, chemistry, and general biology is still largely lacking. In this study, we reexamined 94 Psilocybe fungarium specimens, representing 18 species, by DNA barcoding, evaluated the stability of psilocybin, psilocin, and their related tryptamine alkaloids in 25 specimens across the most commonly vouchered species (Psilocybe cubensis, Psilocybe cyanescens, and Psilocybe semilanceata), and explored the metabolome of cultivated P. cubensis. Our data show that, apart from a few well-known species, the taxonomic accuracy of specimen determinations is largely unreliable, even at the genus level. A substantial quantity of poor-quality and mislabeled sequence data in public repositories, as well as a paucity of sequences derived from types, further exacerbates the problem. Our data also support taxon- and time-dependent decay of psilocybin and psilocin, with some specimens having no detectable quantities of them. We also show that the P. cubensis metabolome possibly contains thousands of uncharacterized compounds, at least some of which may be bioactive. Taken together, our study undermines commonly held assumptions about the accuracy of names and presence of controlled substances in fungarium specimens identified as Psilocybe spp. and reveals that our understanding of the chemical diversity of these mushrooms is largely incomplete. These results have broader implications for regulatory policies pertaining to the storage and sharing of fungarium specimens as well as the use of psychoactive mushrooms for recreation and therapy. IMPORTANCE The therapeutic use of psilocybin, the active ingredient in "magic mushrooms," is revolutionizing mental health care for a number of conditions, including depression, posttraumatic stress disorder (PTSD), and end-of-life care. This has spotlighted the current state of knowledge of psilocybin, including the organisms that endogenously produce it. However, because of international regulation of psilocybin as a controlled substance (often included on the same list as cocaine and heroin), basic research has lagged far behind. Our study highlights how the poor state of knowledge of even the most fundamental scientific information can impact the use of psilocybin-containing mushrooms for recreational or therapeutic applications and undermines critical assumptions that underpin their regulation by legal authorities. Our study shows that currently available chemical studies are mainly inaccurate, irreproducible, and inconsistent, that there exists a high rate of misidentification in museum collections and public databases rendering even names unreliable, and that the concentration of psilocybin and its tryptamine derivatives in three of the most commonly collected Psilocybe species (P. cubensis, P. cyanescens, and P. semilanceata) is highly variable and unstable in museum specimens spanning multiple decades, and our study generates the first-ever insight into the highly complex and largely uncharacterized metabolomic profile for the most commonly cultivated magic mushroom, P. cubensis.

Identification and Characterization of a Cryptic Bifunctional Type I Diterpene Synthase Involved in Talaronoid Biosynthesis from a Marine-Derived Fungus.

We report the identification of the tnd biosynthetic cluster from the marine-derived fungus Aspergillus flavipes and the in vivo characterization of a cryptic type I diterpene synthase. The heterologous expression of the bifunctional terpene synthase led to the discovery of a diterpene backbone, talarodiene, harboring a benzo[a]cyclopenta[d]cyclooctane tricyclic fused ring system. The conversion of geranylgeranyl diphosphate to talarodiene was investigated using 13C-labeling studies, and stable isotope tracer experiments showed the biotransformation of talarodiene into talaronoid C.

Structural Basis of Stereospecific Vanadium-Dependent Haloperoxidase Family Enzymes in Napyradiomycin Biosynthesis.

Vanadium-dependent haloperoxidases (VHPOs) from Streptomyces bacteria differ from their counterparts in fungi, macroalgae, and other bacteria by catalyzing organohalogenating reactions with strict regiochemical and stereochemical control. While this group of enzymes collectively uses hydrogen peroxide to oxidize halides for incorporation into electron-rich organic molecules, the mechanism for the controlled transfer of highly reactive chloronium ions in the biosynthesis of napyradiomycin and merochlorin antibiotics sets the Streptomyces vanadium-dependent chloroperoxidases apart. Here we report high-resolution crystal structures of two homologous VHPO family members associated with napyradiomycin biosynthesis, NapH1 and NapH3, that catalyze distinctive chemical reactions in the construction of meroterpenoid natural products. The structures, combined with site-directed mutagenesis and intact protein mass spectrometry studies, afforded a mechanistic model for the asymmetric alkene and arene chlorination reactions catalyzed by NapH1 and the isomerase activity catalyzed by NapH3. A key lysine residue in NapH1 situated between the coordinated vanadate and the putative substrate binding pocket was shown to be essential for catalysis. This observation suggested the involvement of the ε-NH2, possibly through formation of a transient chloramine, as the chlorinating species much as proposed in structurally distinct flavin-dependent halogenases. Unexpectedly, NapH3 is modified post-translationally by phosphorylation of an active site His (τ-pHis) consistent with its repurposed halogenation-independent, α-hydroxyketone isomerase activity. These structural studies deepen our understanding of the mechanistic underpinnings of VHPO enzymes and their evolution as enantioselective biocatalysts.

A Community Effort: Combining Functional Amplicon Sequencing and Metagenomics Reveals Potential Biosynthetic Gene Clusters Associated with Protective Phenotypes in Rhizosphere Microbiomes.

Soil-dwelling microorganisms associated with plant roots carry out essential processes that promote plant growth and productivity. In addition to these beneficial functions, the rhizosphere microbiome also serves as the first line of defense against many plant pathogens. While many rhizobacteria are capable of producing antifungal natural products, fungal pathogens, such as those belonging to the genus Fusarium, continue to be a major threat to agricultural crops worldwide. In this issue, Tracanna and coworkers (V. Tracanna, A. Ossowicki, M. L. C. Petrus, S. Overduin, et al., mSystems 6:e01116-20, 2021, https://doi.org/10.1128/mSystems.01116-20) implement a targeted amplicon sequencing approach to identify conserved domains and specific metabolic pathways shared among soil samples with antagonistic activities against Fusarium culmorum. They also introduce dom2BGC, an open-source annotation platform that builds co-occurrence networks of natural product-associated domains across samples and aids in putative gene cluster reconstruction. When coupled with metagenomics, functional amplicon sequencing and the dom2BGC pipeline can aid in identifying mechanisms and potential metabolites associated with particular microbiome-associated phenotypes.

Azetidine-Bearing Non-Ribosomal Peptides, Bonnevillamides D and E, Isolated from a Carrion Beetle-Associated Actinomycete.

Two new nonribosomal peptides, bonnevillamides D and E (1 and 2), have been discovered in Streptomyces sp. UTZ13 isolated from the carrion beetle, Nicrophorus concolor. Combinational analysis of the UV, MS, and NMR spectroscopic data revealed that their planar structures were comprised of dichlorinated linear peptides containing nonproteinogenic amino acid residues, such as 4-methylazetidinecarboxylic acid and 4-O-acetyl-5-methylproline. The configurations of bonnevillamides D and E (1 and 2) were determined based on ROESY correlations, the advanced Marfey's method, phenylglycine methyl ester derivatization, molecular modeling, and circular dichroism spectroscopy. The nonribosomal peptide synthetase biosynthetic pathway of bonnevillamides D and E has been proposed using bioinformatic analysis of the whole-genome sequence data of Streptomyces sp. UTZ13. Their biological activity toward the aggregation of amyloid-ß, which is one of the key pathogenic proteins in Alzheimer's disease, was evaluated using a thioflavin T assay and gel electrophoresis. Bonnevillamides D and E reversed the fibril formation by inducing the monomerization of amyloid-ß aggregates.

Marinoterpins A-C: Rare Linear Merosesterterpenoids from Marine-Derived Actinomycete Bacteria of the Family Streptomycetaceae.

The chemical examination of two undescribed marine actinobacteria has yielded three rare merosesterterpenoids, marinoterpins A-C (1-3, respectively). These compounds were isolated from the culture broth extracts of two marine-derived actinomycetes associated with the family Streptomycetaceae, (our strains were CNQ-253 and AJS-327). The structures of the new compounds were determined by extensive interpretation of 1D and 2D NMR, MS, and combined spectroscopic data. These compounds represent new chemical motifs, combining quinoline-N-oxides with a linear sesterterpenoid side chain. Additionally, consistent in all three metabolites is the rare occurrence of two five-ring ethers, which were derived from an apparent cyclization of methyl group carbons to adjacent hydroxy-bearing methylene groups in the sesterterpenoid side chain. Genome scanning of AJS-327 allowed for the identification of the marinoterpin (mrt) biosynthetic cluster, which consists of 16 open-reading frames that code for a sesterterpene pyrophosphate synthase, prenyltransferase, type II polyketide synthase, anthranilate:CoA-ligase, and several tailoring enzymes apparently responsible for installing the N-oxide and bis-tetrahydrofuran ring motifs.

Emerimicins V-X, 15-Residue Peptaibols Discovered from an Acremonium sp. through Integrated Genomic and Chemical Approaches.

Fermentation of Acremonium tubakii W. Gams isolated from a soil sample collected from the University of Utah led to the isolation and characterization of six new linear pentadecapeptides, emerimicins V-X (1-6). Peptaibols containing 15-residues are quite rare, with only 22 reported. Genome mining and bioinformatic analysis were used to identify the emerimicin 60 kbp eme biosynthetic cluster harboring a single 16-module hybrid polyketide-nonribosomal peptide synthetase. A detailed bioinformatic investigation of the corresponding 15 adenylation domains, combined with 1D and 2D NMR experiments, LC-MS/MS data, and advanced Marfey's method, allowed for the elucidation and absolute configuration of all proteinogenic and nonproteinogenic amino acid residues in 1-6. As some peptaibols possess cytotoxic activity, a zebrafish embryotoxicity assay was used to evaluate the toxicity of the six emerimicins and showed that emerimicin V (1) and VI (2) exhibit the most potent activity. Additionally, out of the six emerimicins, 1 displayed modest activity against Enterococcus faecalis, methicillin-resistant Staphylococcus aureus, and vancomycin-resistant Enterococcus faecium with MIC values of 64, 32, and 64 µg/mL, respectively.

Antifungal natural products.

Natural products are specialized small molecules produced in Nature and play pivotal roles in many cellular processes. These compounds possess exquisite chemical diversity and represent some of the most important pharmaceutical agents in human health care. With the rampant rise of fungal pathogens that are becoming resistant to nearly all clinically available antibiotics, there is an increased urgency to find new antifungal therapies with novel modes of action. To meet this need, we must be able to quickly identify new bioactive chemical scaffolds within complex natural extracts, determine their mechanisms of action, and generate appreciable yields for preclinical studies. In this review, we will highlight naturally derived antifungal agents of clinical importance as well as those with strong potential as leads in drug development.

Discovery and characterization of a cytochalasan biosynthetic cluster from the marine-derived fungus Aspergillus flavipes CNL-338.

Cytochalasans are a large family of well-studied cytotoxic molecules isolated from fungi. Investigation into the organic extract of the marine-derived fungal strain Aspergillus flavipes CNL-338 led to the isolation of seven leucine-containing cytochalasans. Genome mining allowed for the identification of the ffs biosynthetic gene cluster, and genetic inactivation studies verified its involvement in cytochalasan biosynthesis. In addition, comparative analysis of key residues in the binding pocket of core cytochalasan biosynthetic enzymes revealed significant similarities among fungal adenylation domains despite differences in substrate preference. We report the identification of leucine-containing cytochalasans from the marine-derived A. flavipes CNL-338 and the characterization of the ffs biosynthetic cluster as verified by genetic inactivation studies.

Complementary Genomic, Bioinformatics, and Chemical Approaches Facilitate the Absolute Structure Assignment of Ionostatin, a Linear Polyketide from a Rare Marine-Derived Actinomycete.

A new linear type-1 polyketide, ionostatin (1), has been fully defined using a combined genomic and bioinformatics approach coupled with confirmatory chemical analyses. The 41 carbon-containing polyether is the product of the 101 kbp ion biosynthetic cluster containing seven modular type-1 polyketide synthases. Ionostatin is composed of 15 chiral centers that were proposed using the stereospecificities installed by the different classes of ketoreductases and enoylreductases and confirmed by rigorous NMR analyses. Incorporated into the structure are two tetrahydrofuran rings that appear to be the product of stereospecific epoxidation, followed by stereospecific ring opening and cyclization. These transformations are proposed to be catalyzed by conserved enzymes analogous to those found in other bacterial-derived polyether biosynthetic clusters. Ionostatin shows moderate cancer cell cytotoxicity against U87 glioblastoma and SKOV3 ovarian carcinoma at 7.4 µg/mL.