Terretonin, ophiobolin, and drimane terpenes with absolute configurations from an algicolous Aspergillus ustus

One new meroterpene, 1,2-dihydroterretonin F (2), five new sesterterpenes, (6a)-21-deoxyophiobolin G(3), (6a)-16,17-dihydro-21-deoxyophiobolin G (4), phiobolin U (5), ophiobolin V (6), and ophiobolin W (7),two new sesquiterpenes, (6-strobilactone-B) esters of (E,E)-6,7-epoxy-2,4-octadienoic acids (13 and 14),and twelve known terpenes (1, 8–12, and 15–20) were isolated from Aspergillus ustus, a fungus from the fresh tissue of marine green alga Codium fragile. Their structures and absolute configurations were identified by NMR and mass spectroscopic methods as well as quantum chemical calculations. Some of the isolates exhibited antibacterial activity and brine shrimp toxicity.One new meroterpene, 1,2-dihydroterretonin F (2), five new sesterterpenes, (6 alpha)-21-deoxyophiobolin G (3), (6 alpha)-16,17-dihydro-21-deoxyophiobolin G (4), ophiobolin U (5), ophiobolin V (6), and ophiobolin W(7), two new sesquiterpenes, (6-strobilactone-B) esters of (E,E)-6,7-epoxy-2,4-octadienoic acids (13 and 14), and twelve known terpenes (1, 8-12, and 15-20) were isolated from Aspergillus ustus, a fungus from the fresh tissue of marine green alga Codium fragile. Their structures and absolute configurations were identified by NMR and mass spectroscopic methods as well as quantum chemical calculations. Some of the isolates exhibited antibacterial activity and brine shrimp toxicity

Building Natural Product Libraries Using Quantitative Clade-Based and Chemical Clustering Strategies

The success of natural product-based drug discovery is predicated on having chemical collections that offer broad coverage of metabolite diversity. We propose a simple set of tools combining genetic barcoding and metabolomics to help investigators build natural product libraries aimed at achieving predetermined levels of chemical coverage. It was found that such tools aided in identifying overlooked pockets of chemical diversity within taxa, which could be useful for refocusing collection strategies. We have used fungal isolates identified as Alternaria from a citizen-science-based soil collection to demonstrate the application of these tools for assessing and carrying out predictive measurements of chemical diversity in a natural product collection. Within Alternaria, different subclades were found to contain nonequivalent levels of chemical diversity. It was also determined that a surprisingly modest number of isolates (195 isolates) was sufficient to afford nearly 99% of Alternaria chemical features in the data set. However, this result must be considered in the context that 17.9% of chemical features appeared in single isolates, suggesting that fungi like Alternaria might be engaged in an ongoing process of actively exploring nature’s metabolic landscape. Our results demonstrate that combining modest investments in securing internal transcribed spacer (ITS)-based sequence information (i.e., establishing gene-based clades) with data from liquid chromatography-mass spectrometry (i.e., generating feature accumulation curves) offers a useful route to obtaining actionable insights into chemical diversity coverage trends in a natural product library. It is anticipated that these outcomes could be used to improve opportunities for accessing bioactive molecules that serve as the cornerstone of natural product-based drug discovery.Open Access fees paid for in whole or in part by the University of Oklahoma Libraries.Ye

Secondary Metabolites from an Algicolous Aspergillus versicolor Strain

Two new compounds, asperversin A (1) and 9ξ-O-2(2,3-dimethylbut-3-enyl)brevianamide Q (2), and nine known compounds, brevianamide K (3), brevianamide M (4), aversin (5), 6,8-di-O-methylnidurufin (6), 6,8-di-O-methylaverufin (7), 6-O-methylaverufin (8), 5α,8α-epidioxyergosta-6,22-dien-3β-ol (9), ergosta-7,22-diene-3β,5α,6β-triol (10), and 6β-methoxyergosta-7,22-diene-3β,5α-diol (11), were obtained from the culture of Aspergillus versicolor, an endophytic fungus isolated from the marine brown alga Sargassum thunbergii. The structures of these compounds were established by spectroscopic techniques. Compounds 4, 7 and 8 exhibited antibacterial activities against Escherichia coli and Staphyloccocus aureus, and 7 also showed lethality against brine shrimp (Artemia salina) with an LC50 value of 0.5 μg/mL

Local Phenomena Shape Backyard Soil Metabolite Composition

Soil covers most of Earth’s continental surface and is fundamental to life-sustaining processes such as agriculture. Given its rich biodiversity, soil is also a major source for natural product drug discovery from soil microorganisms. However, the study of the soil small molecule profile has been challenging due to the complexity and heterogeneity of this matrix. In this study, we implemented high-resolution liquid chromatography–tandem mass spectrometry and large-scale data analysis tools such as molecular networking to characterize the relative contributions of city, state and regional processes on backyard soil metabolite composition, in 188 soil samples collected from 14 USA States, representing five USA climate regions. We observed that region, state and city of collection all influence the overall soil metabolite profile. However, many metabolites were only detected in unique sites, indicating that uniquely local phenomena also influence the backyard soil environment, with both human-derived and naturally-produced (plant-derived, microbially-derived) metabolites identified. Overall, these findings are helping to define the processes that shape the backyard soil metabolite composition, while also highlighting the need for expanded metabolomic studies of this complex environment.This research was supported by start-up funds from the University of Oklahoma (to L.-I.M.). Open Access fees paid for in whole or in part by the University of Oklahoma Libraries.Ye

Opportunistic Sampling of Roadkill as an Entry Point to Accessing Natural Products Assembled by Bacteria Associated with Non-anthropoidal Mammalian Microbiomes

Few secondary metabolites have been reported from mammalian microbiome bacteria despite the large numbers of diverse taxa that inhabit warm-blooded higher vertebrates. As a means to investigate natural products from these microorganisms, an opportunistic sampling protocol was developed, which focused on exploring bacteria isolated from roadkill mammals. This initiative was made possible through the establishment of a newly created discovery pipeline, which couples laser ablation electrospray ionization mass spectrometry (LAESIMS) with bioassay testing, to target biologically active metabolites from microbiome-associated bacteria. To illustrate this process, this report focuses on samples obtained from the ear of a roadkill opossum (Dideiphis virginiana) as the source of two bacterial isolates (Pseudomonas sp. and Serratia sp.) that produced several new and known cyclic lipodepsipeptides (viscosin and serrawettins, respectively). These natural products inhibited biofilm formation by the human pathogenic yeast Candida albicans at concentrations well below those required to inhibit yeast viability. Phylogenetic analysis of 16S rRNA gene sequence libraries revealed the presence of diverse microbial communities associated with different sites throughout the opossum carcass. A putative biosynthetic pathway responsible for the production of the new serrawettin analogues was identified by sequencing the genome of the Serratia sp. isolate. This study provides a functional roadmap to carrying out the systematic investigation of the genomic, microbiological, and chemical parameters related to the production of natural products made by bacteria associated with non-anthropoidal mammalian microbiomes. Discoveries emerging from these studies are anticipated to provide a working framework for efforts aimed at augmenting microbiomes to deliver beneficial natural products to a host.Research reported in this publication was supported by the National Institutes of Health, National Institute of Allergy and Infectious Diseases (1R21AI101487) (R.H.C. and B.S.S.). X-ray data were collected by L. Thomas in the OU Macromolecular Crystallography Laboratory, which is supported, in part, by an Institutional Development Award from the National Institutes of Health, General Medical Sciences (P20GM103640). The C. albicans SC5314 culture was kindly provided by A. Dongari-Bagtzoglou, University of Connecticut Health Center. The LC-ESIMS instrument used for this project was funded in part by a Challenge Grant from the Office of the Vice President for Research, University of Oklahoma, Norman Campus, and an award through the Shimadzu Equipment Grant Program (R.H.C.).Ye

Receptor Heteromerization Expands the Repertoire of Cannabinoid Signaling in Rodent Neurons

A fundamental question in G protein coupled receptor biology is how a single ligand acting at a specific receptor is able to induce a range of signaling that results in a variety of physiological responses. We focused on Type 1 cannabinoid receptor (CB1R) as a model GPCR involved in a variety of processes spanning from analgesia and euphoria to neuronal development, survival and differentiation. We examined receptor dimerization as a possible mechanism underlying expanded signaling responses by a single ligand and focused on interactions between CB1R and delta opioid receptor (DOR). Using co-immunoprecipitation assays as well as analysis of changes in receptor subcellular localization upon co-expression, we show that CB1R and DOR form receptor heteromers. We find that heteromerization affects receptor signaling since the potency of the CB1R ligand to stimulate G-protein activity is increased in the absence of DOR, suggesting that the decrease in CB1R activity in the presence of DOR could, at least in part, be due to heteromerization. We also find that the decrease in activity is associated with enhanced PLC-dependent recruitment of arrestin3 to the CB1R-DOR complex, suggesting that interaction with DOR enhances arrestin-mediated CB1R desensitization. Additionally, presence of DOR facilitates signaling via a new CB1R-mediated anti-apoptotic pathway leading to enhanced neuronal survival. Taken together, these results support a role for CB1R-DOR heteromerization in diversification of endocannabinoid signaling and highlight the importance of heteromer-directed signal trafficking in enhancing the repertoire of GPCR signaling

Genomic and Metabolomic Insights into the Natural Product Biosynthetic Diversity of a Feral-Hog-Associated Brevibacillus laterosporus Strain

The authors thank C. A. Mitchell for advice concerning the organization of the biosynthetic gene clusters in B. laterosporus PE36. We acknowledge J. Villemarete for providing access to the feral hog for sampling. Author contributions Conceived and designed the experiments: BSS RHC. Performed the experiments: CMT BWS JBK LSLP DRP. Analyzed the data: CMT BWS JBK LSLP DRP BSS RHC. Contributed reagents/materials/analysis tools: CMT BWS DRP. Wrote the paper: CMT BWS BSS RHC.Bacteria associated with mammals are a rich source of microbial biodiversity; however, little is known concerning the abilities of these microbes to generate secondary metabolites. This report focuses on a bacterium isolated from the ear of a feral hog from southwestern Oklahoma, USA. The bacterium was identified as a new strain (PE36) of Brevibacillus latersporus, which was shown via genomic analysis to contain a large number of gene clusters presumably involved in secondary metabolite biosynthesis. A scale-up culture of B. latersporus PE36 yielded three bioactive compounds that inhibited the growth of methicillin-resistant Staphylococcus aureus (basiliskamides A and B and 12-methyltetradecanoic acid). Further studies of the isolate's secondary metabolome provided both new (auripyrazine) and previously-described pyrazine-containing compounds. In addition, a new peptidic natural product (auriporcine) was purified that was determined to be composed of a polyketide unit, two L-proline residues, two D-leucine residues, one L-leucine residue, and a reduced L-phenylalanine (L-phenylalanol). An examination of the genome revealed two gene clusters that are likely responsible for generating the basiliskamides and auriporcine. These combined genomic and chemical studies confirm that new and unusual secondary metabolites can be obtained from the bacterial associates of wild mammals.Ye

A Taxonomically-informed Mass Spectrometry Search Tool for Microbial Metabolomics Data

MicrobeMASST, a taxonomically-informed mass spectrometry (MS) search tool, tackles limited microbial metabolite annotation in untargeted metabolomics experiments. Leveraging a curated database of >60,000 microbial monocultures, users can search known and unknown MS/MS spectra and link them to their respective microbial producers via MS/MS fragmentation patterns. Identification of microbial-derived metabolites and relative producers, without a priori knowledge, will vastly enhance the understanding of microorganisms’ role in ecology and human health