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ABSTRACTAims The aim of this research was to isolate oral bacteria that are dependent for growth on adjacent bacteria producing a required growth factor and to identify the chemical structure of the growth factor.Methods Porphyromonas pasteri strain KLE1280, could be cultivated with Staphylococcus hominis and Escherichia coli as helper strains. A deletion mutant library of E. coli was screened to determine genes involved in production of the growth factor. Compounds produced by the growth factor’s pathway were screened to see if they would stimulate growth of strain P. pasteri KLE1280. The genomes of species related to P. pasteri KLE1280 were screened for presence of the factor’s synthetic pathway.Results Analysis of the E. coli deletion mutant library and growth studies identified 1,2-dihydroxy-2-naphthoic acid (DHNA) and menaquinone-4 (MK4) as the growth factors. Strain P. pasteri KLE1280 was shown to lack five genes in the menaquinone synthesis pathway but to possess the two genes necessary to convert DHNA to menaquinone. Genome analysis found that 8 species in genera Porphyromonas and Tannerella lack five genes in the menaquinone synthesis pathway.Conclusions Addition of DHNA to culture media allows isolation of strains of several oral species that are not recovered using standard media

New treatments for the diseases caused by apicomplexans are needed. Recently, we determined that tartrolon E (trtE), a secondary metabolite derived from a shipworm symbiotic bacterium, has broad-spectrum anti-apicomplexan parasite activity. TrtE inhibits apicomplexans at nM concentrations in vitro, including Cryptosporidium parvum, Toxoplasma gondii, Sarcocystis neurona, Plasmodium falciparum, Babesia spp. and Theileria equi. To investigate the mechanism of action of trtE against apicomplexan parasites, we examined changes in the transcriptome of trtE-treated T. gondii parasites. RNA-Seq data revealed that the gene, TGGT1_272370, which is broadly conserved in the coccidia, is significantly upregulated within 4 h of treatment. Using bioinformatics and proteome data available on ToxoDB, we determined that the protein product of this tartrolon E responsive gene (trg) has multiple transmembrane domains, a phosphorylation site, and localizes to the plasma membrane. Deletion of trg in a luciferase-expressing T. gondii strain by CRISPR/Cas9 resulted in a 68% increase in parasite resistance to trtE treatment, supporting a role for the trg protein product in the response of T. gondii to trtE treatment. Trg is conserved in the coccidia, but not in more distantly related apicomplexans, indicating that this response to trtE may be unique to the coccidians, and other mechanisms may be operating in other trtE-sensitive apicomplexans. Uncovering the mechanisms by which trtE inhibits apicomplexans may identify shared pathways critical to apicomplexan parasite survival and advance the search for new treatments.

Terpenoids are the largest and structurally most diverse class of natural products. They possess potent and specific biological activity in multiple assays and against diseases, including cancer and malaria as notable examples. Although the number of characterized terpenoid molecules is huge, our knowledge of how they are biosynthesized is limited, particularly when compared to the well-studied thiotemplate assembly lines. Bacteria have only recently been recognized as having the genetic potential to biosynthesize a large number of complex terpenoids, but our current ability to associate genetic potential with molecular structure is severely restricted. The canonical terpene biosynthetic pathway uses a single enzyme to form a cyclized hydrocarbon backbone followed by modifications with a suite of tailoring enzymes that can generate dozens of different products from a single backbone. This functional promiscuity of terpene biosynthetic pathways renders terpene biosynthesis susceptible to rational pathway engineering using the latest developments in the field of synthetic biology. These engineered pathways will not only facilitate the rational creation of both known and novel terpenoids, their development will deepen our understanding of a significant branch of biosynthesis. The biosynthetic insights gained will likely empower a greater degree of engineering proficiency for non-natural terpene biosynthetic pathways and pave the way towards the biotechnological production of high value terpenoids.

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.

Chemical investigations of a marine sponge-associated Bacillus revealed six new imidazolium-containing compounds, bacillimidazoles A–F (1–6). Previous reports of related imidazolium-containing natural products are rare. Initially unveiled by timsTOF (trapped ion mobility spectrometry) MS data, extensive HRMS and 1D and 2D NMR analyses enabled the structural elucidation of 1–6. In addition, a plausible biosynthetic pathway to bacillimidazoles is proposed based on isotopic labeling experiments and invokes the highly reactive glycolytic adduct 2,3-butanedione. Combined, the results of structure elucidation efforts, isotopic labeling studies and bioinformatics suggest that 1–6 result from a fascinating intersection of primary and secondary metabolic pathways in Bacillus sp. WMMC1349. Antimicrobial assays revealed that, of 1–6, only compound six displayed discernible antibacterial activity, despite the close structural similarities shared by all six natural products.

Symbiotic microbes help a myriad of insects acquire nutrients. Recent work suggests that insects also frequently associate with actinobacterial symbionts that produce molecules to help defend against parasites and predators. Here we explore a potential association between Actinobacteria and two species of fungus-farming ambrosia beetles, Xyleborinus saxesenii and Xyleborus affinis. We isolated and identified actinobacterial and fungal symbionts from laboratory reared nests, and characterized small molecules produced by the putative actinobacterial symbionts. One 16S rRNA phylotype of Streptomyces (XylebKG-1) was abundantly and consistently isolated from the galleries and adults of X. saxesenii and X. affinis nests. In addition to Raffaelea sulphurea, the symbiont that X. saxesenii cultivates, we also repeatedly isolated a strain of Nectria sp. that is an antagonist of this mutualism. Inhibition bioassays between Streptomyces griseus XylebKG-1 and the fungal symbionts from X. saxesenii revealed strong inhibitory activity of the actinobacterium toward the fungal antagonist Nectria sp. but not the fungal mutualist R. sulphurea. Bioassay guided HPLC fractionation of S. griseus XylebKG-1 culture extracts, followed by NMR and mass spectrometry, identified cycloheximide as the compound responsible for the observed growth inhibition. A biosynthetic gene cluster putatively encoding cycloheximide was also identified in S. griseus XylebKG-1. The consistent isolation of a single 16S phylotype of Streptomyces from two species of ambrosia beetles, and our finding that a representative isolate of this phylotype produces cycloheximide, which inhibits a parasite of the system but not the cultivated fungus, suggests that these actinobacteria may play defensive roles within these systems.

Apicomplexan parasites cause severe disease in both humans and their domesticated animals. Since these parasites readily develop drug resistance, development of new, effective drugs to treat infection caused by these parasites is an ongoing challenge for the medical and veterinary communities. We hypothesized that invertebrate-bacterial symbioses might be a rich source of anti-apicomplexan compounds because invertebrates are susceptible to infections with gregarines, parasites that are ancestral to all apicomplexans. We chose to explore the therapeutic potential of shipworm symbiotic bacteria as they are bona fide symbionts, are easily grown in axenic culture and have genomes rich in secondary metabolite loci [1,2]. Two strains of the shipworm symbiotic bacterium, Teredinibacter turnerae, were screened for activity against Toxoplasma gondii and one strain, T7901, exhibited activity against intracellular stages of the parasite. Bioassay-guided fractionation identified tartrolon E (trtE) as the source of the activity. TrtE has an EC50 of 3 nM against T. gondii, acts directly on the parasite itself and kills the parasites after two hours of treatment. TrtE exhibits nanomolar to picomolar level activity against Cryptosporidium, Plasmodium, Babesia, Theileria, and Sarcocystis; parasites representing all branches of the apicomplexan phylogenetic tree. The compound also proved effective against Cryptosporidium parvum infection in neonatal mice, indicating that trtE may be a potential lead compound for preclinical development. Identification of a promising new compound after such limited screening strongly encourages further mining of invertebrate symbionts for new anti-parasitic therapeutics.

Abstract Background The human gut microbiome has been linked to numerous components of health and disease. However, approximately 25% of the bacterial species in the gut remain uncultured, which limits our ability to properly understand, and exploit, the human microbiome. Previously, we found that growing environmental bacteria in situ in a diffusion chamber enables growth of uncultured species, suggesting the existence of growth factors in the natural environment not found in traditional cultivation media. One source of growth factors proved to be neighboring bacteria, and by using co-culture, we isolated previously uncultured organisms from the marine environment and identified siderophores as a major class of bacterial growth factors. Here, we employ similar co-culture techniques to grow bacteria from the human gut microbiome and identify novel growth factors. Results By testing dependence of slow-growing colonies on faster-growing neighboring bacteria in a co-culture assay, eight taxonomically diverse pairs of bacteria were identified, in which an “induced” isolate formed a gradient of growth around a cultivatable “helper.” This set included two novel species Faecalibacterium sp. KLE1255—belonging to the anti-inflammatory Faecalibacterium genus—and Sutterella sp. KLE1607. While multiple helper strains were identified, Escherichia coli was also capable of promoting growth of all induced isolates. Screening a knockout library of E. coli showed that a menaquinone biosynthesis pathway was required for growth induction of Faecalibacterium sp. KLE1255 and other induced isolates. Purified menaquinones induced growth of 7/8 of the isolated strains, quinone specificity profiles for individual bacteria were identified, and genome analysis suggests an incomplete menaquinone biosynthetic capability yet the presence of anaerobic terminal reductases in the induced strains, indicating an ability to respire anaerobically. Conclusions Our data show that menaquinones are a major class of growth factors for bacteria from the human gut microbiome. These organisms are taxonomically diverse, including members of the genus Faecalibacterium, Bacteroides, Bilophila, Gordonibacter, and Sutterella. This suggests that loss of quinone biosynthesis happened independently in many lineages of the human microbiota. Quinones can be used to improve existing bacterial growth media or modulate the human gut microbiota by encouraging the growth of important symbionts, such as Faecalibacterium species.

Some anaerobic bacteria use insoluble minerals as terminal electron acceptors and discovering the ways in which electrons move through the membrane barrier to the exterior acceptor forms an active field of research with implications for both bacterial physiology and bioenergy. A previous study suggested that Shewanella oneidensis MR-1 utilizes a small, polar, redox active molecule that serves as an electron shuttle between the bacteria and insoluble acceptors, but the shuttle itself has never been identified. Through isolation and synthesis, we identify it as ACNQ (2-amino-3-carboxy-1,4-naphthoquinone), a soluble analog of menaquinone. ACNQ is derived from DHNA (1,4-dihydroxy-2-naphthoic acid) in a non-enzymatic process that frustrated genetic approaches to identify the shuttle. Both ACNQ and DHNA restore reduction of AQDS under anaerobic growth in menaquinone-deficient mutants. Bioelectrochemistry analyses reveal that ACNQ (−0.32 VAg/AgCl) contributes to the extracellular electron transfer (EET) as an electron shuttle, without altering menaquinone generation or EET related cytochrome c expression.

The Brazilian stingless bee Scaptotrigona depilis requires the brood cells-associated fungus Zygosaccharomyces sp. as steroid source for metamorphosis. Besides the presence of Zygosaccharomyces sp., other fungi inhabit S. depilis brood cells, but their biological functions are unknown. Here we show that Candida sp. and Monascus ruber, isolated from cerumen of S. depilis brood provisions, interact with Zygosaccharomyces sp. and modulate its growth. Candida sp. produces volatile organic compounds (VOCs) that stimulate Zygosacchromyces sp. development. Monascus ruber inhibits Zygosacchromyces sp. growth by producing lovastatin, which blocks steroid biosynthesis. We also observed that in co-cultures M. ruber inhibits Candida sp. through the production of monascin. The modulation of Zygosaccharomyces sp. growth by brood cell-associated fungi suggests their involvement in S. depilis larval development. This tripartite fungal community opens new perspectives in the research of microbial interactions with bees.

Enteric Gram-negative rods (GNR), which are frequent causes of community-acquired and nosocomial infections, are increasingly resistant to the antibiotics in our current armamentarium. One solution to this medical dilemma is the development of novel classes of antimicrobial compounds. Here we report the development of a robust, whole cell-based, high-throughput metabolic assay that detects compounds with activity against carbapenem-resistant Klebsiella pneumoniae. We have used this assay to screen approximately 8,000 fungal extracts and 50,000 synthetic compounds with the goal of identifying extracts and compounds active against a highly resistant strain of Klebsiella pneumoniae. The primary screen identified 43 active fungal extracts and 144 active synthetic compounds. Patulin, a known fungal metabolite and inhibitor of bacterial quorum sensing and alanine racemase, was identified as the active component in the most potent fungal extracts. We did not study patulin further due to previously published evidence of toxicity. Three synthetic compounds termed O06, C17, and N08 were chosen for further study. Compound O06 did not have significant antibacterial activity but rather interfered with sugar metabolism, while compound C17 had only moderate activity against GNRs. Compound N08 was active against several resistant GNRs and showed minimal toxicity to mammalian cells. Preliminary studies suggested that it interferes with protein expression. However, its direct application may be limited by susceptibility to efflux and a tendency to form aggregates in aqueous media. Rapid screening of 58,000 test samples with identification of several compounds that act on CR-K. pneumoniae demonstrates the utility of this screen for the discovery of drugs active against this highly resistant GNR.

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.

Robust preference for fermentative glucose metabolism has motivated domestication of the budding yeast Saccharomyces cerevisiae. This program can be circumvented by a protein-based genetic element, the [GAR+] prion, permitting simultaneous metabolism of glucose and other carbon sources. Diverse bacteria can elicit yeast cells to acquire [GAR+], although the molecular details of this interaction remain unknown. Here we identify the common bacterial metabolite lactic acid as a strong [GAR+] inducer. Transient exposure to lactic acid caused yeast cells to heritably circumvent glucose repression. This trait had the defining genetic properties of [GAR+], and did not require utilization of lactic acid as a carbon source. Lactic acid also induced [GAR+]-like epigenetic states in fungi that diverged from S. cerevisiae ~200 million years ago, and in which glucose repression evolved independently. To our knowledge, this is the first study to uncover a bacterial metabolite with the capacity to potently induce a prion.

Deficiency of mitochondrial complex I is encountered in both rare and common diseases, but we have limited therapeutic options to treat this lesion to the oxidative phosphorylation system (OXPHOS). Idebenone and menadione are redox-active molecules capable of rescuing OXPHOS activity by engaging complex I-independent pathways of entry, often referred to as "complex I bypass." In the present study, we created a cellular model of complex I deficiency by using CRISPR genome editing to knock out Ndufa9 in mouse myoblasts, and utilized this cell line to develop a high-throughput screening platform for novel complex I bypass factors. We screened a library of ~40,000 natural product extracts and performed bioassay-guided fractionation on a subset of the top scoring hits. We isolated four plant-derived 1,4-naphthoquinone complex I bypass factors with structural similarity to menadione: chimaphilin and 3-chloro-chimaphilin from Chimaphila umbellata and dehydro-α-lapachone and dehydroiso-α-lapachone from Stereospermum euphoroides. We also tested a small number of structurally related naphthoquinones from commercial sources and identified two additional compounds with complex I bypass activity: 2-methoxy-1,4-naphthoquinone and 2-methoxy-3-methyl-1,4,-naphthoquinone. The six novel complex I bypass factors reported here expand this class of molecules and will be useful as tool compounds for investigating complex I disease biology.

In previous work, we characterized the strong neuroprotective properties of the marine compound Psammaplysene A (PA) in in vitro and in vivo models of neurodegeneration. Based on its strong neuroprotective activity, the current work attempts to identify the physical target of PA to gain mechanistic insight into its molecular action. Two distinct methods, used in parallel, to purify protein-binding partners of PA led to the identification of HNRNPK as a direct target of PA. Based on surface plasmon resonance, we find that the binding of PA to HNRNPK is RNA-dependent. These findings suggest a role for HNRNPK-dependent processes in neurodegeneration/neuroprotection, and warrant further study of HNRNPK in this context.

While the human gut microbiota are suspected to produce diffusible small molecules that modulate host signaling pathways, few of these molecules have been identified. Species of Bacteroides and their relatives, which often comprise >50% of the gut community, are unusual among bacteria in that their membrane is rich in sphingolipids, a class of signaling molecules that play a key role in inducing apoptosis and modulating the host immune response. Although known for more than three decades, the full repertoire of Bacteroides sphingolipids has not been defined. Here, we use a combination of genetics and chemistry to identify the sphingolipids produced by Bacteroides fragilis NCTC 9343. We constructed a deletion mutant of BF2461, a putative serine palmitoyltransferase whose yeast homolog catalyzes the committed step in sphingolipid biosynthesis. We show that the Δ2461 mutant is sphingolipid deficient, enabling us to purify and solve the structures of three alkaline-stable lipids present in the wild-type strain but absent from the mutant. The first compound was the known sphingolipid ceramide phosphorylethanolamine, and the second was its corresponding dihydroceramide base. Unexpectedly, the third compound was the glycosphingolipid α-galactosylceramide (α-GalCer(Bf)), which is structurally related to a sponge-derived sphingolipid (α-GalCer, KRN7000) that is the prototypical agonist of CD1d-restricted natural killer T (iNKT) cells. We demonstrate that α-GalCer(Bf) has similar immunological properties to KRN7000: it binds to CD1d and activates both mouse and human iNKT cells both in vitro and in vivo. Thus, our study reveals BF2461 as the first known member of the Bacteroides sphingolipid pathway, and it indicates that the committed steps of the Bacteroides and eukaryotic sphingolipid pathways are identical. Moreover, our data suggest that some Bacteroides sphingolipids might influence host immune homeostasis.

A chemoproteomics approach has been employed to identify a kinase that could be used as a druggable target in efforts to develop new treatments for African sleeping sickness.

In sexually reproducing animals, mating is essential for transmitting genetic information to the next generation and therefore animals have evolved mechanisms for optimizing the chance of successful mate location. In the soil nematode C. elegans, males approach hermaphrodites via the ascaroside pheromones, recognize hermaphrodites when their tails contact the hermaphrodites' body, and eventually mate with them. These processes are mediated by sensory signals specialized for sexual communication, but other mechanisms may also be used to optimize mate location. Here we describe associative learning whereby males use sodium chloride as a cue for hermaphrodite location. Both males and hermaphrodites normally avoid sodium chloride after associative conditioning with salt and starvation. However, we found that males become attracted to sodium chloride after conditioning with salt and starvation if hermaphrodites are present during conditioning. For this conditioning, which we call sexual conditioning, hermaphrodites are detected by males through pheromonal signaling and additional cue(s). Sex transformation experiments suggest that neuronal sex of males is essential for sexual conditioning. Altogether, these results suggest that C. elegans males integrate environmental, internal and social signals to determine the optimal strategy for mate location.

Topoisomerase inhibitors are effective for antibacterial and anticancer therapy because they can lead to the accumulation of the intermediate DNA cleavage complex formed by the topoisomerase enzymes, which trigger cell death. Here we report the application of a novel enzyme-based high-throughput screening assay to identify natural product extracts that can lead to increased accumulation of the DNA cleavage complex formed by recombinant Yersinia pestis topoisomerase I as part of a larger effort to identify new antibacterial compounds. Further characterization and fractionation of the screening positives from the primary assay led to the discovery of a depside, anziaic acid, from the lichen Hypotrachyna sp. as an inhibitor for both Y. pestis and Escherichia coli topoisomerase I. In in vitro assays, anziaic acid exhibits antibacterial activity against Bacillus subtilis and a membrane permeable strain of E. coli. Anziaic acid was also found to act as an inhibitor of human topoisomerase II but had little effect on human topoisomerase I. This is the first report of a depside with activity as a topoisomerase poison inhibitor and demonstrates the potential of this class of natural products as a source for new antibacterial and anticancer compounds.

ABSTRACT Burkholderia thailandensis produces a family of polyketide-peptide molecules called bactobolins, some of which are potent antibiotics. We found that growth of B. thailandensis at 30°C versus that at 37°C resulted in increased production of bactobolins. We purified the three most abundant bactobolins and determined their activities against a battery of bacteria and mouse fibroblasts. Two of the three compounds showed strong activities against both bacteria and fibroblasts. The third analog was much less potent in both assays. These results suggested that the target of bactobolins might be conserved across bacteria and mammalian cells. To learn about the mechanism of bactobolin activity, we isolated four spontaneous bactobolin-resistant Bacillus subtilis mutants. We used genomic sequencing technology to show that each of the four resistant variants had mutations in rplB, which codes for the 50S ribosome-associated L2 protein. Ectopic expression of a mutant rplB gene in wild-type B. subtilis conferred bactobolin resistance. Finally, the L2 mutations did not confer resistance to other antibiotics known to interfere with ribosome function. Our data indicate that bactobolins target the L2 protein or a nearby site and that this is not the target of other antibiotics. We presume that the mammalian target of bactobolins involves the eukaryotic homolog of L2 (L8e). IMPORTANCE Currently available antibiotics target surprisingly few cellular functions, and there is a need to identify novel antibiotic targets. We have been interested in the Burkholderia thailandensis bactobolins, and we sought to learn about the target of bactobolin activity by mapping spontaneous resistance mutations in the bactobolin-sensitive Bacillus subtilis. Our results indicate that the bactobolin target is the 50S ribosome-associated L2 protein or a region of the ribosome affected by L2. Bactobolin-resistant mutants are not resistant to other known ribosome inhibitors. Our evidence indicates that bactobolins interact with a novel antibiotic target.

Bacterially-produced small molecules exert profound influences on animal health, morphogenesis, and evolution through poorly understood mechanisms. In one of the closest living relatives of animals, the choanoflagellate Salpingoeca rosetta, we find that rosette colony development is induced by the prey bacterium Algoriphagus machipongonensis and its close relatives in the Bacteroidetes phylum. Here we show that a rosette inducing factor (RIF-1) produced by A. machipongonensis belongs to the small class of sulfonolipids, obscure relatives of the better known sphingolipids that play important roles in signal transmission in plants, animals, and fungi. RIF-1 has extraordinary potency (femtomolar, or 10−15 M) and S. rosetta can respond to it over a broad dynamic range—nine orders of magnitude. This study provides a prototypical example of bacterial sulfonolipids triggering eukaryotic morphogenesis and suggests molecular mechanisms through which bacteria may have contributed to the evolution of animals.

Due to the inexorable invasion of our hospitals and communities by drug-resistant bacteria, there is a pressing need for novel antibacterial agents. Here we report the development of a sensitive and robust but low-tech and inexpensive high-throughput metabolic screen for novel antibiotics. This screen is based on a colorimetric assay of pH that identifies inhibitors of bacterial sugar fermentation. After validation of the method, we screened over 39,000 crude extracts derived from organisms that grow in the diverse ecosystems of Costa Rica and identified 49 with reproducible antibacterial effects. An extract from an endophytic fungus was further characterized, and this led to the discovery of three novel natural products. One of these, which we named mirandamycin, has broad-spectrum antibacterial activity against Escherichia coli, Pseudomonas aeruginosa, Vibrio cholerae, methicillin-resistant Staphylococcus aureus, and Mycobacterium tuberculosis. This demonstrates the power of simple high throughput screens for rapid identification of new antibacterial agents from environmental samples.

Oridonin is a diterpenoid with anti-cancer activity that occurs in the Chinese medicinal plant Isodon rubescens and some related species. While the bioactivity of oridonin has been well studied, the extent of natural variation in the production of this compound is poorly known. This study characterizes natural variation in oridonin production in order to guide selection of populations of Isodon with highest oridonin yield. Different populations of I. rubescens and related species were collected in China, and their offspring were grown in a greenhouse. Samples were examined for oridonin content, genotyped using 11 microsatellites, and representatives were sequenced for three phylogenetic markers (ITS, rps16, trnL-trnF). Oridonin production was mapped on a molecular phylogeny of the genus Isodon using samples from each population as well as previously published Genbank sequences. Oridonin has been reported in 12 out of 74 species of Isodon examined for diterpenoids, and the phylogeny indicates that oridonin production has arisen at least three times in the genus. Oridonin production was surprisingly consistent between wild-collected parents and greenhouse-grown offspring, despite evidence of gene flow between oridonin-producing and non-producing populations of Isodon. Additionally, microsatellite genetic distance between individuals was significantly correlated with chemical distance in both parents and offspring. Neither heritability nor correlation with genetic distance were significant when the comparison was restricted to only populations of I. rubescens, but this result should be corroborated using additional samples. Based on these results, future screening of Isodon populations for oridonin yield should initially prioritize a broad survey of all species known to produce oridonin, rather than focusing on multiple populations of one species, such as I. rubescens. Of the samples examined here, I. rubescens or I. japonicus from Henan province would provide the best source of oridonin.

Bacteria produce small molecule iron chelators, known as siderophores, to facilitate the acquisition of iron from the environment. The synthesis of more than one siderophore and the production of multiple siderophore uptake systems by a single bacterial species are common place. The selective advantages conferred by the multiplicity of siderophore synthesis remains poorly understood. However, there is growing evidence suggesting that siderophores may have other physiological roles besides their involvement in iron acquisition.Here we provide the first report that pyochelin displays antibiotic activity against some bacterial strains. Observation of differential sensitivity to pyochelin against a panel of bacteria provided the first indications that catecholate siderophores, produced by some bacteria, may have roles other than iron acquisition. A pattern emerged where only those strains able to make catecholate-type siderophores were resistant to pyochelin. We were able to associate pyochelin resistance to catecholate production by showing that pyochelin-resistant Escherichia coli became sensitive when biosynthesis of its catecholate siderophore enterobactin was impaired. As expected, supplementation with enterobactin conferred pyochelin resistance to the entE mutant. We observed that pyochelin-induced growth inhibition was independent of iron availability and was prevented by addition of the reducing agent ascorbic acid or by anaerobic incubation. Addition of pyochelin to E. coli increased the levels of reactive oxygen species (ROS) while addition of ascorbic acid or enterobactin reduced them. In contrast, addition of the carboxylate-type siderophore, citrate, did not prevent pyochelin-induced ROS increases and their associated toxicity.We have shown that the catecholate siderophore enterobactin protects E. coli against the toxic effects of pyochelin by reducing ROS. Thus, it appears that catecholate siderophores can behave as protectors of oxidative stress. These results support the idea that siderophores can have physiological roles aside from those in iron acquisition.

Identifying new sources for small molecule discovery is necessary to help mitigate the continuous emergence of antibiotic-resistance in pathogenic microbes. Recent studies indicate that one potentially rich source of novel natural products is Actinobacterial symbionts associated with social and solitary Hymenoptera. Here we test this possibility by examining two species of solitary mud dauber wasps, Sceliphron caementarium and Chalybion californicum. We performed enrichment isolations from 33 wasps and obtained more than 200 isolates of Streptomyces Actinobacteria. Chemical analyses of 15 of these isolates identified 11 distinct and structurally diverse secondary metabolites, including a novel polyunsaturated and polyoxygenated macrocyclic lactam, which we name sceliphrolactam. By pairing the 15 Streptomyces strains against a collection of fungi and bacteria, we document their antifungal and antibacterial activity. The prevalence and anti-microbial properties of Actinobacteria associated with these two solitary wasp species suggest the potential role of these Streptomyces as antibiotic-producing symbionts, potentially helping defend their wasp hosts from pathogenic microbes. Finding phylogenetically diverse and chemically prolific Actinobacteria from solitary wasps suggests that insect-associated Actinobacteria can provide a valuable source of novel natural products of pharmaceutical interest.

Transient receptor potential (TRP) ion channels mediate key aspects of taste, smell, pain, temperature sensation, and pheromone detection. To deepen our understanding of TRP channel physiology, we require more diverse pharmacological tools. Citral, a bioactive component of lemongrass, is commonly used as a taste enhancer, as an odorant in perfumes, and as an insect repellent. Here we report that citral activates TRP channels found in sensory neurons (TRPV1 and TRPV3, TRPM8, and TRPA1), and produces long-lasting inhibition of TRPV1-3 and TRPM8, while transiently blocking TRPV4 and TRPA1. Sustained citral inhibition is independent of internal calcium concentration, but is state-dependent, developing only after TRP channel opening. Citral's actions as a partial agonist are not due to cysteine modification of the channels nor are they a consequence of citral's stereoisoforms. The isolated aldehyde and alcohol cis and trans enantiomers (neral, nerol, geranial, and geraniol) each reproduce citral's actions. In juvenile rat dorsal root ganglion neurons, prolonged citral inhibition of native TRPV1 channels enabled the separation of TRPV2 and TRPV3 currents. We find that TRPV2 and TRPV3 channels are present in a high proportion of these neurons (94% respond to 2-aminoethyldiphenyl borate), consistent with our immunolabeling experiments and previous in situ hybridization studies. The TRPV1 activation requires residues in transmembrane segments two through four of the voltage-sensor domain, a region previously implicated in capsaicin activation of TRPV1 and analogous menthol activation of TRPM8. Citral's broad spectrum and prolonged sensory inhibition may prove more useful than capsaicin for allodynia, itch, or other types of pain involving superficial sensory nerves and skin.