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In nature, halogenation is a strategy used to increase the biological activity of secondary metabolites, compounds that are often effective as drugs. However, halides are not particularly reactive unless they are activated, typically by oxidation. The pace of discovery of new enzymes for halogenation is increasing, revealing new metalloenzymes, flavoenzymes, S -adenosyl- L -methionine (SAM)-dependent enzymes and others that catalyse halide oxidation using dioxygen, hydrogen peroxide and hydroperoxides, or that promote nucleophilic halide addition reactions.

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.

Hepatocellular carcinoma (HCC) represents the third leading cause of cancer-related death worldwide and has been increasing in developed nations. The MYC oncogene or its paralogs are frequently amplified or overexpressed in subtypes of cancer associated with stem cell-like features and worse clinical outcomes, including in liver cancer. Unfortunately, selective inhibitors that target MYC or its transcriptional program are not yet clinically available for therapy of HCC. Here, we identified methionine metabolism as a selective vulnerability for MYC but not RAS-driven liver cancers. MYC-driven liver cancer cells are methionine dependent, with markedly diminished tumor growth when mice are fed a methionine low diet. While RAS-driven liver cancer was resistant to a low methionine diet. S-adenosylmethionine (SAM), the predominant methyl donor, partially rescues cell proliferation following methionine depletion, suggesting that methylation processes are especially critical in the context of MYC high tumor cells. Heavy isotope methionine tracing in MYC high cells identified increased levels of m5C nucleotides. We found NOP2, an rRNA m5C-methyltransferase, was regulated by both MYC overexpression and methionine abundance linking the two processes. Methionine depletion reduced methylation of multiple 28S rRNA residues as did NOP2 knockdown. Depletion of NOP2 selectively inhibited MYC liver cancer cell proliferation and tumor growth. Thus, methionine catabolism is critical for MYC-driven liver tumorigenesis and the rRNA methyltransferase NOP2 may serve as a new therapeutic target in liver cancer.

Core Ideas Leaf fungal symbionts represent a potential new tool in agriculture. Effective fungal application requires an understanding of their interactions. Fungal interactions resulted in non‐additive plant growth and wilt responses. Fungal metabolites indicated qualitative additive, synergistic, or antagonistic plant responses. Fungal trait dissimilarity predicted the size of plant response deviations. Fungal symbionts are increasingly targeted as tools for crop management, but their use in the field requires an understanding of how fungi interact in a community context. Fungal interactions may result in additive effects on the host plant, which could be predicted simply based on individual fungal behavior. Alternatively, interactions among fungi may result in non‐additive synergistic or antagonistic effects on plant performance that are more challenging to predict. Here, we hypothesized that the effects of fungal interactions on the plant host could be predicted from their niche overlap. To test this idea, we used foliar fungal endophytes with a range of niche overlap based on trait dissimilarities to examine the effects of six fungal species pairs compared to the corresponding individual fungal species on switchgrass (Panicum virgatum L.) in water‐stressed and well‐watered conditions. Mixtures of endophytes had either no effect or predictable, additive effects on plant tiller number, but effects on plant growth rate and wilting were largely non‐additive. Moisture level, fungal stress, and metabolic trait dissimilarity predicted 51 to 92% of the deviation of fungal effects from additive, with less similar fungi likely to have more synergistic effects on the plant host. Furthermore, we identified indicator metabolites for fungal interaction outcomes. However, the effects of endophyte interactions on the plant host were environment dependent making single community applications more challenging. Overall, future development of microbial tools for use in agriculture must consider their interactions to optimize application.

Pharmaceuticals have extensive reciprocal interactions with the microbiome, but whether bacterial drug sensitivity and metabolism is driven by pathways conserved in host cells remains unclear. Here we show that anti-cancer fluoropyrimidine drugs inhibit the growth of gut bacterial strains from 6 phyla. In both Escherichia coli and mammalian cells, fluoropyrimidines disrupt pyrimidine metabolism. Proteobacteria and Firmicutes metabolized 5-fluorouracil to its inactive metabolite dihydrofluorouracil, mimicking the major host mechanism for drug clearance. The preTA operon was necessary and sufficient for 5-fluorouracil inactivation by E. coli , exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice and was prevalent in the gut microbiomes of colorectal cancer patients. The conservation of both the targets and enzymes for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug efficacy and side-effect profiles. Anti-cancer fluoropyrimidine drugs have antibacterial effects on the gut microbiome, and these drugs can be metabolized by gut bacteria via conserved pathways also found in mammalian hosts.

Fungal symbionts living inside plant leaves (“endophytes”) can vary from beneficial to parasitic, but the mechanisms by which the fungi affect the plant host phenotype remain poorly understood. Chemical interactions are likely the proximal mechanism of interaction between foliar endophytes and the plant, as individual fungal strains are often exploited for their diverse secondary metabolite production. Here, we go beyond single strains to examine commonalities in how 16 fungal endophytes shift plant phenotypic traits such as growth and physiology, and how those relate to plant metabolomics profiles. We inoculated individual fungi on switchgrass, Panicum virgatum L. This created a limited range of plant growth and physiology (2–370% of fungus-free controls on average), but effects of most fungi overlapped, indicating functional similarities in unstressed conditions. Overall plant metabolomics profiles included almost 2000 metabolites, which were broadly correlated with plant traits across all the fungal treatments. Terpenoid-rich samples were associated with larger, more physiologically active plants and phenolic-rich samples were associated with smaller, less active plants. Only 47 metabolites were enriched in plants inoculated with fungi relative to fungus-free controls, and of these, Lasso regression identified 12 metabolites that explained from 14 to 43% of plant trait variation. Fungal long-chain fatty acids and sterol precursors were positively associated with plant photosynthesis, conductance, and shoot biomass, but negatively associated with survival. The phytohormone gibberellin, in contrast, was negatively associated with plant physiology and biomass. These results can inform ongoing efforts to develop metabolites as crop management tools, either by direct application or via breeding, by identifying how associations with more beneficial components of the microbiome may be affected.

Pharmaceuticals are the top predictor of inter-individual variations in gut microbial community structure1, consistent with in vitro evidence that host-targeted drugs inhibit gut bacterial growth2 and are extensively metabolized by the gut microbiome3,4. In oncology, bacterial metabolism has been implicated in both drug efficacy5,6 and toxicity7,8; however, the degree to which bacterial drug sensitivity and metabolism can be driven by conserved pathways also found in mammalian cells remains poorly understood. Here, we show that anticancer fluoropyrimidine drugs inhibit the growth of diverse gut bacterial strains by disrupting pyrimidine metabolism, as in mammalian cells. Select bacteria metabolized 5-fluorouracil (5-FU) to its inactive metabolite dihydrofluorouracil (DHFU), mimicking the major host pathway for drug clearance. The preTA operon was necessary and sufficient for 5-FU inactivation in Escherichia coli, exhibited high catalytic efficiency for the reductive reaction, decreased the bioavailability and efficacy of oral fluoropyrimidine treatment in mice, and was prevalent in the gut microbiomes of colorectal cancer patients prior to and during treatment. The observed conservation of both the targets and pathways for metabolism of therapeutics across domains highlights the need to distinguish the relative contributions of human and microbial cells to drug disposition9, efficacy, and side effect profiles. Competing Interest Statement P.J.T. is on the scientific advisory boards for Kaleido, Pendulum, Seed, and SNIPRbiome; there is no direct overlap between the current study and these consulting duties. K.S.P. is on the scientific advisory board for Phylagen; there is no direct overlap between the current study and these consulting duties. C.E.A serves on the scientific advisory board for Pionyr Immunotherapeutics and has received research funding (institution) from Bristol Meyer Squibb, Guardant Health, Kura Oncology, Merck, and Novartis; there is no direct overlap with the current study. All other authors have no relevant declarations. Footnotes * Major revisions to main text and figures.

Shipworms are marine bivalve mollusks (Family Teredinidae) that use wood for shelter and food. They harbor a group of closely related, yet phylogenetically distinct, bacterial endosymbionts in bacteriocytes located in the gills. This endosymbiotic community is believed to support the host's nutrition in multiple ways, through the production of cellulolytic enzymes and the fixation of nitrogen. The genome of the shipworm endosymbiont Teredinibacter turnerae T7901 was recently sequenced and in addition to the potential for cellulolytic enzymes and diazotrophy, the genome also revealed a rich potential for secondary metabolites. With nine distinct biosynthetic gene clusters, nearly 7% of the genome is dedicated to secondary metabolites. Bioinformatic analyses predict that one of the gene clusters is responsible for the production of a catecholate siderophore. Here we describe this gene cluster in detail and present the siderophore product from this cluster. Genes similar to the entCEBA genes of enterobactin biosynthesis involved in the production and activation of dihydroxybenzoic acid (DHB) are present in this cluster, as well as a two-module non-ribosomal peptide synthetase (NRPS). A novel triscatecholate siderophore, turnerbactin, was isolated from the supernatant of iron-limited T. turnerae T7901 cultures. Turnerbactin is a trimer of N-(2,3-DHB)-L-Orn-L-Ser with the three monomeric units linked by Ser ester linkages. A monomer, dimer, dehydrated dimer, and dehydrated trimer of 2,3-DHB-L-Orn-L-Ser were also found in the supernatant. A link between the gene cluster and siderophore product was made by constructing a NRPS mutant, TtAH03. Siderophores could not be detected in cultures of TtAH03 by HPLC analysis and Fe-binding activity of culture supernatant was significantly reduced. Regulation of the pathway by iron is supported by identification of putative Fur box sequences and observation of increased Fe-binding activity under iron restriction. Evidence of a turnerbactin fragment was found in shipworm extracts, suggesting the production of turnerbactin in the symbiosis.

The majority of microorganisms require iron for growth. However, due to the insolubility of iron at neutral pH in aerobic environments, the amount of iron available for bacteria is severely limited. When bacteria are starved for iron, as occurs in the oceans where concentrations of iron are sub-nanomolar, or at the onset of infection in a vertebrate host where iron is locked away by iron storage and transport proteins as an immediate immune response, bacteria rely on the production of siderophores to acquire iron. Siderophores are low molecular weight, organic compounds with high affinity for Fe(III). This dissertation describes the isolation and structure elucidation of several new triscatecholate siderophores produced by different bacteria. The marine bacterium Vibrio campbellii DS40M4 was found to produce a new triscatecholate siderophore, trivanchrobactin, a related new biscatecholamide compound, divanchrobactin, and the previously reported siderophores vanchrobactin and anguibactin. Vanchrobactin is comprised of L-serine, D-arginine, and 2,3-dihydroxybenzoic acid (DHBA), while trivanchrobactin is a linear trimer of vanchrobactin joined by two serine ester linkages. Similarly, the plant pathogen Dickeya chrysanthemi EC16 was found to produce a new triscatecholate siderophore, cyclic trichrysobactin, the related catecholate compounds, linear trichrysobactin and dichrysobactin, and the previously reported monomeric siderophore unit, chrysobactin. Chrysobactin is comprised of L-serine, D-lysine, and 2,3-DHBA. Trichrysobactin is a cyclic trimer of chrysobactin joined by a triserine lactone backbone. Further progress towards the structure characterization of the amphienterobactins, produced by the coastal marine bacterial isolate Vibrio sp. Roatan-5, is also reported herein. In addition, Pseudomonas sp. Zp2, a bacterium isolated from the basalt surface of the underwater volcano Vailulu’u Seamount, was found to produce the known hydroxymate siderophore, desferrioxamine E. Vanadium bromoperoxidase isolated from the marine red alga Delisea pulchra was previously shown to mediate the bromolactonization of 4-pentynoic acid to form 5E-bromomethylidenetetrahydro-2-furanone in the biosynthesis of synthetic halogenated furanones. Reported herein are the effects of 5E-bromomethylidenetetrahydro- 2-furanone, which is structurally related to the naturally occurring bromofuranones isolated from Delisea pulchra, on quorum sensing in Agrobacterium tumefaciens. 5E-bromomethylidenetetrahydro-2-furanone was found to disrupt quorum sensing in Agrobacterium tumefaciens bioassays.