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The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host‐microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host‐microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell‐free time‐resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco‐2) in the aerobic chamber. Caco‐2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host‐microbe coculture, compared to respective mono‐culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'‐monophosphate, asparagine, and thiamine. Additionally, while Caco‐2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3‐dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes. [ABSTRACT FROM AUTHOR]

Type 2 diabetes (T2D) is a global public health issue characterized by excess weight, abdominal obesity, dyslipidemia, hyperglycemia, and a progressive increase in insulin resistance. Human population studies of T2D development and its effects on systemic metabolism are confounded by many factors that cannot be controlled, complicating the interpretation of results and the identification of early biomarkers. Aged, sedentary, and overweight/obese non-human primates (NHPs) are one of the best animal models to mimic spontaneous T2D development in humans. We sought to identify and distinguish a set of plasma and/or fecal metabolite biomarkers, that have earlier disease onset predictability, and that could be evaluated for their predictability in subsequent T2D studies in human cohorts. In this study, a single plasma and fecal sample was collected from each animal in a colony of 57 healthy and dysmetabolic NHPs and analyzed for metabolomics and lipidomics. The samples were comprehensively analyzed using untargeted and targeted LC/MS/MS. The changes in each animal's disease phenotype were monitored using IVGTT, HbA1c, and other clinical metrics, and correlated with their metabolic profile. The plasma and fecal lipids, as well as bile acid profiles, from Healthy, Dysmetabolic (Dys), and Diabetic (Dia) animals were compared. Following univariate and multivariate analyses, including adjustments for weight, age, and sex, several plasma lipid species were identified to be significantly different between these animal groups. Medium and long-chain plasma phosphatidylcholines (PCs) ranked highest at distinguishing Healthy from Dys animals, whereas plasma triglycerides (TG) primarily distinguished Dia from Dys animals. Random Forest (RF) analysis of fecal bile acids showed a reduction in the secondary bile acid glycoconjugate, GCDCA, in diseased animals (AUC 0.76[0.64, 0.89]). Moreover, metagenomics results revealed several bacterial species, belonging to the genera Roseburia, Ruminococcus, Clostridium, and Streptococcus, to be both significantly enriched in non-healthy animals and associated with secondary bile acid levels. In summary, our results highlight the detection of several elevated circulating plasma PCs and microbial species associated with fecal secondary bile acids in NHP dysmetabolic states. The lipids and metabolites we have identified may help researchers to differentiate individual NHPs more precisely between dysmetabolic and overtly diabetic states. This could help assign animals to study groups that are more likely to respond to potential therapies where a difference in efficacy might be anticipated between early vs. advanced disease. [ABSTRACT FROM AUTHOR]

Humans use natural products to treat disease; similarly, some insects use natural products produced by Actinobacteria to combat infectious pathogens. Honey bees, Apis mellifera , are ecologically and economically important for their critical role as plant pollinators and are host to diverse and potentially virulent pathogens that threaten hive health. Here, we provide evidence that Actinobacteria that can suppress pathogenic microbes are associated with A. mellifera. We show through culture-dependent approaches that Actinobacteria in the genus Streptomyces are commonly isolated from foraging bees, and especially common in pollen stores. One strain, isolated from pollen stores, exhibited pronounced inhibitory activity against Paenibacillus larvae , the causative agent of American foulbrood. Bioassay-guided HPLC fractionation, followed by NMR and mass spectrometry, identified the known macrocyclic polyene lactam, piceamycin that was responsible for this activity. Further, we show that in its purified form, piceamycin has potent inhibitory activity toward P. larvae. Our results suggest that honey bees may use pollen-derived Actinobacteria and their associated small molecules to mediate colony health. Given the importance of honey bees to modern agriculture and their heightened susceptibility to disease, the discovery and development of antibiotic compounds from hives could serve as an important strategy in supporting disease management within apiaries. [ABSTRACT FROM AUTHOR]

Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane‐bridge‐containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms. [ABSTRACT FROM AUTHOR]

Rediscovery of known natural products hinders the discovery of new, unique scaffolds. Efforts have mostly focused on streamlining the determination of what compounds are known vs. unknown (dereplication), but an alternative strategy is to focus on what is different. Utilizing statistics and assuming that common actinobacterial metabolites are likely known, focus can be shifted away from dereplication and towards discovery. LC-MS-based principal component analysis (PCA) provides a perfect tool to distinguish unique vs. common metabolites, but the variability inherent within natural products leads to datasets that do not fit ideal standards. To simplify the analysis of PCA models, we developed a script that identifies only those masses or molecules that are unique to each strain within a group, thereby greatly reducing the number of data points to be inspected manually. Since the script is written in R, it facilitates integration with other metabolomics workflows and supports automated mass matching to databases such as Antibase. [ABSTRACT FROM AUTHOR]

Bacterial communities associated with marine invertebrates such as sponges and ascidians have demonstrated potential as sources of bio-medically relevant small molecules. Metagenomic analysis has shown that many of these invertebrates harbor populations of Actinobacteria, many of which are cultivable. While some populations within invertebrates are transmitted vertically, others are obtained from the environment. We hypothesized that cultivable diversity from sponges living in brackish mangrove habitats have associations with Actinobacterial populations that differ from those found in clear tropical waters. In this study, we analyzed the cultivable Actinobacterial populations from sponges found in these two distinct habitats with the aim of understanding the secondary metabolite potential. Importantly, we wanted to broadly evaluate the potential differences among these groups to guide future Actinobacterial collection strategies for the purposes of drug discovery. [ABSTRACT FROM AUTHOR]

Three new dentigerumycin analogues are produced by Streptomyces sp. M41, a bacterium isolated from a South African termite, Macrotermes natalensis. The structures of the complex nonribosomal peptide synthetase-polyketide synthase (NRPS/PKS) hybrid compounds were determined by 1D- and 2D-NMR spectroscopy, high-resolution mass spectrometry, and circular dichroism (CD) spectroscopy. Both cyclic and linear peptides are reported, and the genetic organization of the NRPS modules within the biosynthetic gene cluster accounts for the observed structural diversity. [ABSTRACT FROM AUTHOR]

LipL and Cpr19 are nonheme, mononuclear Fe( II)-dependent, α-ketoglutarate (α KG): UMP oxygenases that catalyze the formation of CO2, succinate, phosphate, and uridine-5′-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes-[1′,3′,4′,5′,5′-2H] UMP and the phosphonate derivative of UMP-and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2H atom of [1′,3′,4′,5′,5′-2H] UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5′ R)-5′-hydroxy- UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases. [ABSTRACT FROM AUTHOR]

Emiliania huxleyi is a model coccolithophore micro-alga that generates vast blooms in the ocean. Bacteria are not considered among the major factors influencing coccolithophore physiology. Here we show through a laboratory model system that the bacterium Phaeobacter inhibens, a well-studied member of the Roseobacter group, intimately interacts with E. huxleyi. While attached to the algal cell, bacteria initially promote algal growth but ultimately kill their algal host. Both algal growth enhancement and algal death are driven by the bacterially-produced phytohormone indole-3-acetic acid. Bacterial production of indole-3-acetic acid and attachment to algae are significantly increased by tryptophan, which is exuded from the algal cell. Algal death triggered by bacteria involves activation of pathways unique to oxidative stress response and programmed cell death. Our observations suggest that bacteria greatly influence the physiology and metabolism of E. huxleyi. Coccolithophore-bacteria interactions should be further studied in the environment to determine whether they impact micro-algal population dynamics on a global scale. [ABSTRACT FROM AUTHOR]

Utilization of 2H, 13C, and 15N isotopically labeled proteins and peptides is now routine in biomolecular NMRinvestigations. The widespread availability of inexpensive, uniformly 13C enriched glucose now makes it possible to isolate uniformly 13C labeled natural products from microbial fermentation. We now wish to describe an approach for the rapid structural characterization of uniformly 13C labeled natural products that avoids the pitfalls of relying on parameters typically employed in biomolecular NMR studies. [ABSTRACT FROM AUTHOR]

Ascidians contain abundant, diverse secondary metabolites, which are thought to serve a defensive role and which have been applied to drug discovery. It is known that bacteria in symbiosis with ascidians produce several of these metabolites, but very little is known about factors governing these 'chemical symbioses'. To examine this phenomenon across a wide geographical and species scale, we performed bacterial and chemical analyses of 32 different ascidians, mostly from the didemnid family from Florida, Southern California and a broad expanse of the tropical Pacific Ocean. Bacterial diversity analysis showed that ascidian microbiomes are highly diverse, and this diversity does not correlate with geographical location or latitude. Within a subset of species, ascidian microbiomes are also stable over time (R=−0.037, P-value=0.499). Ascidian microbiomes and metabolomes contain species-specific and location-specific components. Location-specific bacteria are found in low abundance in the ascidians and mostly represent strains that are widespread. Location-specific metabolites consist largely of lipids, which may reflect differences in water temperature. By contrast, species-specific bacteria are mostly abundant sequenced components of the microbiomes and include secondary metabolite producers as major components. Species-specific chemicals are dominated by secondary metabolites. Together with previous analyses that focused on single ascidian species or symbiont type, these results reveal fundamental properties of secondary metabolic symbiosis. Different ascidian species have established associations with many different bacterial symbionts, including those known to produce toxic chemicals. This implies a strong selection for this property and the independent origin of secondary metabolite-based associations in different ascidian species. The analysis here streamlines the connection of secondary metabolite to producing bacterium, enabling further biological and biotechnological studies. [ABSTRACT FROM AUTHOR]

Fungus-growing ants engage in complex symbiotic relationships with their fungal crop, specialized fungal pathogens, and bacteria that provide chemical defenses. In an effort to understand the evolutionary origins of this multilateral system, we investigated bacteria isolated from fungi. One bacterial strain (Streptomyces sp. CLI2509) from the bracket fungus Hymenochaete rubiginosa, produced an unusual peptide, tryptorubin A, which contains heteroaromatic links between side chains that give it a rigid polycyclic globular structure. The three-dimensional structure was determined by NMR and MS, including a 13C-13C COSY of isotopically enriched material, degradation, derivatives, and computer modeling. Whole genome sequencing identified a likely pair of biosynthetic genes responsible for tryptorubin A’s linear hexapeptide backbone. The genome also revealed the close relationship between CLI2509 and Streptomyces sp. SPB78, which was previously implicated in an insect–bacterium symbiosis.

Forazoline A, a novel antifungal polyketide with in vivo efficacy against Candida albicans, was discovered using LCMS-based metabolomics to investigate marine-invertebrate-associated bacteria. Forazoline A had a highly unusual and unprecedented skeleton. Acquisition of 13C-13C gCOSY and 13C-15N HMQC NMR data provided the direct carbon-carbon and carbon-nitrogen connectivity, respectively. This approach represents the first example of determining direct 13C-15N connectivity for a natural product. Using yeast chemical genomics, we propose that forazoline A operated through a new mechanism of action with a phenotypic outcome of disrupting membrane integrity. [ABSTRACT FROM AUTHOR]

Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions. [ABSTRACT FROM AUTHOR]

Two novel trialkyl-substituted aromatic acids, solwaric acids A and B, were isolated from a marine Solwaraspora sp. cultivated from the ascidian Trididemnum orbiculatum. Solwaric acids A and B were isotopically labeled with U-13C glucose, and analysis of a 13C-13C COSY allowed for unambiguous determination of the location of the phenyl methyl group. The two novel compounds demonstrated antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-sensitive Staphylococcus aureus (MSSA). [ABSTRACT FROM AUTHOR]

Two novel chlorinated pyrones, halomadurones A and B, and two novel brominated analogues, halomadurones C and D, were isolated from a marine Actinomadura sp. cultivated from the ascidian Ecteinascidia turbinata. Additionally, a non-halogenated analogue, 2-methyl-6-((E)-3-methyl-1,3-hexadiene)-γ-pyrone, was synthesized to understand the role of the halogens for activity. Halomadurones C and D demonstrated potent nuclear factor E2-related factor antioxidant response element (Nrf2-ARE) activation, which is an important therapeutic approach for treatment of neurodegenerative diseases. [ABSTRACT FROM AUTHOR]

Microtermolides A (1) and B (2) were isolated from a Streptomyces sp. strain associated with fungus-growing termites. The structures of 1 and 2 were determined by 1D- and 2D-NMR spectroscopy and high-resolution mass spectrometry. Structural elucidation of 1 led to the re-examination of the structure originally proposed for vinylamycin (3). Based on a comparison of predicted and experimental \(^1\)H and \(^{13}\)C NMR chemical shifts, we propose that vinylamycin’s structure be revised from 3 to 4.

A marine Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis was found to produce thiocoraline, a potent cytotoxic compound. Five new analogs of thiocoraline were isolated and represent the first analogs of thiocoraline. 22'-Deoxythiocoraline (2), thiochondrilline C (5), and 12'-sulfoxythiocoraline (6) demonstrated significant cytotoxicity against the A549 human cancer cell line with EC50 values of 0.13, 2.86, and 1.26 µM, respectively. The analogs provide insight into the SAR and biosynthesis of thiocoraline. The DP4 probability method was used to analyze ab initio NMR calculations to confirm stereochemical assignments. [ABSTRACT FROM AUTHOR]

Utilization of isotopically labeled proteins and peptides is a routinely employed approach in biomolecular NMR investigations. The widespread availability of inexpensive, uniformly 13C ‐ enriched glucose now makes it possible to produce uniformly 13C ‐ labeled natural products by microbial fermentation. In this feature article, the authors describe an experimental approach for the rapid structural characterization of uniformly 13C ‐ labeled natural products based on the Constant ‐ Time HSQC (CT ‐ HSQC) experiment. Rigorous theoretical evaluation of the CT ‐ HSQC experiment allowed the applicability of the experiment to be expanded from the traditional, narrow scope of labeled amino acids to encompass virtually any small molecule or U ‐ 13C labeled natural product. A suite of experiments including CT ‐ HSQC, 13C ‐ 13C COSY, and COSYLR experiments is sufficient for the structure elucidation of uniformly 13C ‐ labeled small molecules and natural products. Differences in NMR approaches for structure elucidation of natural abundance and uniformly 13C ‐ labeled molecules are also discussed. The present work provides a researcher working in this area of natural products chemistry with NMR structure elucidation tools for investigating 13C ‐ labeled small molecules and natural products. [ABSTRACT FROM AUTHOR]

Summary The gut microbiota modulate host biology in numerous ways, but little is known about the molecular mediators of these interactions. Previously, we found a widely distributed family of nonribosomal peptide synthetase gene clusters in gut bacteria. Here, by expressing a subset of these clusters in Escherichia coli or Bacillus subtilis , we show that they encode pyrazinones and dihydropyrazinones. At least one of the 47 clusters is present in 88% of the National Institutes of Health Human Microbiome Project (NIH HMP) stool samples, and they are transcribed under conditions of host colonization. We present evidence that the active form of these molecules is the initially released peptide aldehyde, which bears potent protease inhibitory activity and selectively targets a subset of cathepsins in human cell proteomes. Our findings show that an approach combining bioinformatics, synthetic biology, and heterologous gene cluster expression can rapidly expand our knowledge of the metabolic potential of the microbiota while avoiding the challenges of cultivating fastidious commensals. [ABSTRACT FROM AUTHOR]

Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions. [ABSTRACT FROM AUTHOR]

Donor-to-donor variability in primary human organoid cultures has not been well characterized. As these cultures contain multiple cell types, there is greater concern that variability could lead to increased noise. In this work we investigated donor-to-donor variability in human gut adult stem cell (ASC) organoids. We examined intestinal developmental pathways during culture differentiation in ileum- and colon-derived cultures established from multiple donors, showing that differentiation patterns were consistent among cultures. This finding indicates that donor-to-donor variability in this system remains at a manageable level. Intestinal metabolic activity was evaluated by targeted analysis of central carbon metabolites and by analyzing hormone production patterns. Both experiments demonstrated similar metabolic functions among donors. Importantly, this activity reflected intestinal biology, indicating that these ASC organoid cultures are appropriate for studying metabolic processes. This work establishes a framework for generating high-confidence data using human primary cultures through thorough characterization of variability., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tapinarof is a stilbene drug that is used to treat psoriasis and atopic dermatitis, and is thought to function through regulation of the AhR and Nrf2 signaling pathways, which have also been linked to inflammatory bowel diseases. It is produced by the gammaproteobacterial Photorhabdus genus, which thus represents a model to probe tapinarof structural and functional transformations. We show that Photorhabdus transforms tapinarof into novel drug metabolism products that kill inflammatory bacteria, and that a cupin enzyme contributes to the conversion of tapinarof and related dietary stilbenes into novel dimers. One dimer has activity against methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VRE), and another undergoes spontaneous cyclizations to a cyclopropane-bridge-containing hexacyclic framework that exhibits activity against Mycobacterium. These dimers lack efficacy in a colitis mouse model, whereas the monomer reduces disease symptoms., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Escherichia coli is a common inhabitant of the human microbiota and a beacon model organism in biology. However, an understanding of its signaling systems that regulate population-level phenotypes known as quorum sensing remain incomplete. Here, we define the structure and biosynthesis of autoinducer-3 (AI-3), a metabolite of previously unknown structure involved in the pathogenesis of enterohemorrhagic E. coli (EHEC). We demonstrate that novel AI-3 analogs are derived from threonine dehydrogenase (Tdh) products and "abortive" tRNA synthetase reactions, and they are distributed across a variety of Gram-negative and Gram-positive bacterial pathogens. In addition to regulating virulence genes in EHEC, we show that the metabolites exert diverse immunological effects on primary human tissues. The discovery of AI-3 metabolites and their biochemical origins now provides a molecular foundation for investigating the diverse biological roles of these elusive yet widely distributed bacterial signaling molecules., Competing Interests: The authors declare no competing financial interest., (Copyright © 2020 American Chemical Society.)

Forazoline A is a structurally complex PKS-NRPS hybrid produced by marine-derived Actinomadura sp. During the course of studies highlighting the application of IFS analysis as a powerful tool for natural products analysis, we were alerted to an earlier misinterpretation with respect to forazoline A structure elucidation. In particular, IFS reveals that forazoline A contains a thioketone moiety rarely seen in secondary metabolites and, thus, constitutes an even more intriguing structure than originally thought.

Chemistry drives many biological interactions between the microbiota and host animals, yet it is often challenging to identify the chemicals involved. This poses a problem, as such small molecules are excellent sources of potential pharmaceuticals, pretested by nature for animal compatibility. We discovered anti-HIV compounds from small, marine tunicates from the Eastern Fields of Papua New Guinea. Tunicates are a reservoir for new bioactive chemicals, yet their small size often impedes identification or even detection of the chemicals within. We solved this problem by combining chemistry, metagenomics, and synthetic biology to directly identify and synthesize the natural products. We show that these anti-HIV compounds, the divamides, are a novel family of lanthipeptides produced by symbiotic bacteria living in the tunicate. Neighboring animal colonies contain structurally related divamides that differ starkly in their biological properties, suggesting a role for biosynthetic plasticity in a native context wherein biological interactions take place.

A polyether antibiotic, ecteinamycin (1), was isolated from a marine Actinomadura sp., cultivated from the ascidian Ecteinascidia turbinata. 13 C enrichment, high resolution NMR spectroscopy, and molecular modeling enabled elucidation of the structure of 1, which was validated on the basis of comparisons with its recently reported crystal structure. Importantly, ecteinamycin demonstrated potent activity against the toxigenic strain of Clostridium difficile NAP1/B1/027 (MIC = 59 ng/μL), as well as other toxigenic and nontoxigenic C. difficile isolates both in vitro and in vivo. Additionally, chemical genomics studies using Escherichia coli barcoded deletion mutants led to the identification of sensitive mutants such as trkA and kdpD involved in potassium cation transport and homeostasis supporting a mechanistic proposal that ecteinamycin acts as an ionophore antibiotic. This is the first antibacterial agent whose mechanism of action has been studied using E. coli chemical genomics. On the basis of these data, we propose ecteinamycin as an ionophore antibiotic that causes C. difficile detoxification and cell death via potassium transport dysregulation.

Establishing the relative configuration of a bioactive natural product represents the most challenging part in determining its structure. Residual dipolar couplings (RDCs) are sensitive probes of the relative spatial orientation of internuclear vectors. We adapted a force field structure calculation methodology to allow free sampling of both R and S configurations of the stereocenters of interest. The algorithm uses a floating alignment tensor in a simulated annealing protocol to identify the conformations and configurations that best fit experimental RDC and distance restraints (from NOE and J-coupling data). A unique configuration (for rigid molecules) or a very small number of configurations (for less rigid molecules) of the structural models having the lowest chiral angle energies and reasonable magnitudes of the alignment tensor are provided as the best predictions of the unknown configuration. For highly flexible molecules, the progressive locking of their stereocenters into their statistically dominant R or S state dramatically reduces the number of possible relative configurations. The result is verified by checking that the same configuration is obtained by initiating the locking from different regions of the molecule. For all molecules tested having known configurations (with conformations ranging from mostly rigid to highly flexible), the method accurately determined the correct configuration.

Natural products (secondary metabolites) found in marine invertebrates are often thought to be produced by resident symbiotic bacteria, and these products appear to play a major role in the symbiotic interaction of bacteria and their hosts. In these animals, there is extensive variation, both in chemistry and in the symbiotic bacteria that produce them. Here, we sought to answer the question of what factors underlie chemical variation in the ocean. As a model, we investigated the colonial tunicate Lissoclinum patella because of its rich and varied chemistry and its broad geographic range. We sequenced mitochondrial cytochrome c oxidase 1 (COXI) genes, and found that animals classified as L. patella fall into three phylogenetic groups that may encompass several cryptic species. The presence of individual natural products followed the phylogenetic relationship of the host animals, even though the compounds are produced by symbiotic bacteria that do not follow host phylogeny. In sum, we show that cryptic populations of animals underlie the observed chemical diversity, suggesting that the host controls selection for particular secondary metabolite pathways. These results imply novel approaches to obtain chemical diversity from the oceans, and also demonstrate that the diversity of marine natural products may be greatly impacted by cryptic local extinctions.

Medullary thyroid cancer (MTC) is an aggressive neuroendocrine tumor (NET). Previous research has shown that activation of Notch signaling has a tumor suppressor role in NETs. The potential therapeutic effect of thiocoraline on the activation of the Notch pathway in an MTC cell line (TT) was investigated. Thiocoraline was isolated from a marine bacterium Verrucosispora sp. MTT assay (3-[4, 5-dimethylthiazole-2-yl]-2, 5-diphenyltetrazolium bromide) was used to determine the IC50 value and to measure cell proliferation. Western blot revealed the expression of Notch isoforms, NET, and cell cycle markers. Cell cycle progression was validated by flow cytometry. The mRNA expression of Notch isoforms and downstream targets were measured using real-time PCR. The IC50 value for thiocoraline treatment in TT cells was determined to be 7.6 nmol/L. Thiocoraline treatment decreased cell proliferation in a dose- and time-dependent manner. The mechanism of growth inhibition was found to be cell cycle arrest in G1 phase. Thiocoraline activated the Notch pathway as demonstrated by the dose-dependent increase in mRNA and protein expression of Notch isoforms. Furthermore, treatment with thiocoraline resulted in changes in the expression of downstream targets of the Notch pathway (HES1, HES2, HES6, HEY1, and HEY2) and reduced expression of NET markers, CgA, and ASCL1. Thiocoraline is a potent Notch pathway activator and an inhibitor of MTC-TT cell proliferation at low nanomolar concentrations. These results provide exciting evidence for the use of thiocoraline as a potential treatment for intractable MTC., (© 2013 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Natural products profoundly impact many research areas, including medicine, organic chemistry, and cell biology. However, discovery of new natural products suffers from a lack of high throughput analytical techniques capable of identifying structural novelty in the face of a high degree of chemical redundancy. Methods to select bacterial strains for drug discovery have historically been based on phenotypic qualities or genetic differences and have not been based on laboratory production of secondary metabolites. Therefore, untargeted LC/MS-based secondary metabolomics was evaluated to rapidly and efficiently analyze marine-derived bacterial natural products using LC/MS-principal component analysis (PCA). A major goal of this work was to demonstrate that LC/MS-PCA was effective for strain prioritization in a drug discovery program. As proof of concept, we evaluated LC/MS-PCA for strain selection to support drug discovery, for the discovery of unique natural products, and for rapid assessment of regulation of natural product production.

A marine Nocardia sp. isolated from the ascidian Trididemnum orbiculatum was found to produce five new lipopeptides, peptidolipins B-F (1-5), which show distinct similarities to the previously reported L-Val(6) analog of peptidolipin NA. Synthetic modification of peptidolipin E (4) was used to determine the location of an olefin within the lipid chain. The advanced Marfey's method was used to determine the absolute configurations of the amino acids. Peptidolipins B (1) and E (4) demonstrated moderate antibacterial activity against methicillin-resistant Staphylococcus aureus and methicillin-sensitive Staphylococcus aureus., (© 2012 American Chemical Society and American Society of Pharmacognosy)

We recently developed a 2D 1H-13C HSQC0 approach to quantify individual chemicals in complex mixtures. The HSQC0 approach has been implemented in phase-cycled and gradient-selective versions. As in quantitative 1D NMR, the normalized integrated signal intensities in HSQC0 are proportional to the concentrations of individual chemicals in the mixture. We applied the HSQC0 approach to selectively quantify thiocoraline present at a level of 1% w/w in an extract from a Verrucosispora sp. isolated from the sponge Chondrilla caribensis f. caribensis. We expect that this approach can be used to quantify other natural products of interest in extracts without prior purification.