Tundrenone: An Atypical Secondary Metabolite from Bacteria with Highly Restricted Primary Metabolism

Methane-oxidizing bacteria, aerobes that utilize methane as their sole carbon and energy source, are being increasingly studied for their environmentally significant ability to remove methane from the atmosphere. Their genomes indicate that they also have a robust and unusual secondary metabolism. Bioinformatic analysis of the Methylobacter tundripaludum genome identified biosynthetic gene clusters for several intriguing metabolites, and this report discloses the structural and genetic characterization of tundrenone, one of these metabolites. Tundrenone is a highly oxidized metabolite that incorporates both a modified bicyclic chorismate-derived fragment and a modified lipid tail bearing a β,γ-unsaturated α-hydroxy ketone. Tundrenone has been genetically linked to its biosynthetic gene cluster, and quorum sensing activates its production. M. tundripaludum’s genome and tundrenone’s discovery support the idea that additional studies of methane-oxidizing bacteria will reveal new naturally occurring molecular scaffolds and the biosynthetic pathways that produce them

Canvass: a crowd-sourced, natural-product screening library for exploring biological space

NCATS thanks Dingyin Tao for assistance with compound characterization. This research was supported by the Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH). R.B.A. acknowledges support from NSF (CHE-1665145) and NIH (GM126221). M.K.B. acknowledges support from NIH (5R01GM110131). N.Z.B. thanks support from NIGMS, NIH (R01GM114061). J.K.C. acknowledges support from NSF (CHE-1665331). J.C. acknowledges support from the Fogarty International Center, NIH (TW009872). P.A.C. acknowledges support from the National Cancer Institute (NCI), NIH (R01 CA158275), and the NIH/National Institute of Aging (P01 AG012411). N.K.G. acknowledges support from NSF (CHE-1464898). B.C.G. thanks the support of NSF (RUI: 213569), the Camille and Henry Dreyfus Foundation, and the Arnold and Mabel Beckman Foundation. C.C.H. thanks the start-up funds from the Scripps Institution of Oceanography for support. J.N.J. acknowledges support from NIH (GM 063557, GM 084333). A.D.K. thanks the support from NCI, NIH (P01CA125066). D.G.I.K. acknowledges support from the National Center for Complementary and Integrative Health (1 R01 AT008088) and the Fogarty International Center, NIH (U01 TW00313), and gratefully acknowledges courtesies extended by the Government of Madagascar (Ministere des Eaux et Forets). O.K. thanks NIH (R01GM071779) for financial support. T.J.M. acknowledges support from NIH (GM116952). S.M. acknowledges support from NIH (DA045884-01, DA046487-01, AA026949-01), the Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program (W81XWH-17-1-0256), and NCI, NIH, through a Cancer Center Support Grant (P30 CA008748). K.N.M. thanks the California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board for support. B.T.M. thanks Michael Mullowney for his contribution in the isolation, elucidation, and submission of the compounds in this work. P.N. acknowledges support from NIH (R01 GM111476). L.E.O. acknowledges support from NIH (R01-HL25854, R01-GM30859, R0-1-NS-12389). L.E.B., J.K.S., and J.A.P. thank the NIH (R35 GM-118173, R24 GM-111625) for research support. F.R. thanks the American Lebanese Syrian Associated Charities (ALSAC) for financial support. I.S. thanks the University of Oklahoma Startup funds for support. J.T.S. acknowledges support from ACS PRF (53767-ND1) and NSF (CHE-1414298), and thanks Drs. Kellan N. Lamb and Michael J. Di Maso for their synthetic contribution. B.S. acknowledges support from NIH (CA78747, CA106150, GM114353, GM115575). W.S. acknowledges support from NIGMS, NIH (R15GM116032, P30 GM103450), and thanks the University of Arkansas for startup funds and the Arkansas Biosciences Institute (ABI) for seed money. C.R.J.S. acknowledges support from NIH (R01GM121656). D.S.T. thanks the support of NIH (T32 CA062948-Gudas) and PhRMA Foundation to A.L.V., NIH (P41 GM076267) to D.S.T., and CCSG NIH (P30 CA008748) to C.B. Thompson. R.E.T. acknowledges support from NIGMS, NIH (GM129465). R.J.T. thanks the American Cancer Society (RSG-12-253-01-CDD) and NSF (CHE1361173) for support. D.A.V. thanks the Camille and Henry Dreyfus Foundation, the National Science Foundation (CHE-0353662, CHE-1005253, and CHE-1725142), the Beckman Foundation, the Sherman Fairchild Foundation, the John Stauffer Charitable Trust, and the Christian Scholars Foundation for support. J.W. acknowledges support from the American Cancer Society through the Research Scholar Grant (RSG-13-011-01-CDD). W.M.W.acknowledges support from NIGMS, NIH (GM119426), and NSF (CHE1755698). A.Z. acknowledges support from NSF (CHE-1463819). (Intramural Research Program of the National Center for Advancing Translational Sciences, National Institutes of Health (NIH); CHE-1665145 - NSF; CHE-1665331 - NSF; CHE-1464898 - NSF; RUI: 213569 - NSF; CHE-1414298 - NSF; CHE1361173 - NSF; CHE1755698 - NSF; CHE-1463819 - NSF; GM126221 - NIH; 5R01GM110131 - NIH; GM 063557 - NIH; GM 084333 - NIH; R01GM071779 - NIH; GM116952 - NIH; DA045884-01 - NIH; DA046487-01 - NIH; AA026949-01 - NIH; R01 GM111476 - NIH; R01-HL25854 - NIH; R01-GM30859 - NIH; R0-1-NS-12389 - NIH; R35 GM-118173 - NIH; R24 GM-111625 - NIH; CA78747 - NIH; CA106150 - NIH; GM114353 - NIH; GM115575 - NIH; R01GM121656 - NIH; T32 CA062948-Gudas - NIH; P41 GM076267 - NIH; R01GM114061 - NIGMS, NIH; R15GM116032 - NIGMS, NIH; P30 GM103450 - NIGMS, NIH; GM129465 - NIGMS, NIH; GM119426 - NIGMS, NIH; TW009872 - Fogarty International Center, NIH; U01 TW00313 - Fogarty International Center, NIH; R01 CA158275 - National Cancer Institute (NCI), NIH; P01 AG012411 - NIH/National Institute of Aging; Camille and Henry Dreyfus Foundation; Arnold and Mabel Beckman Foundation; Scripps Institution of Oceanography; P01CA125066 - NCI, NIH; 1 R01 AT008088 - National Center for Complementary and Integrative Health; W81XWH-17-1-0256 - Office of the Assistant Secretary of Defense for Health Affairs through the Peer Reviewed Medical Research Program; P30 CA008748 - NCI, NIH, through a Cancer Center Support Grant; California Department of Food and Agriculture Pierce's Disease and Glassy Winged Sharpshooter Board; American Lebanese Syrian Associated Charities (ALSAC); University of Oklahoma Startup funds; 53767-ND1 - ACS PRF; PhRMA Foundation; P30 CA008748 - CCSG NIH; RSG-12-253-01-CDD - American Cancer Society; RSG-13-011-01-CDD - American Cancer Society; CHE-0353662 - National Science Foundation; CHE-1005253 - National Science Foundation; CHE-1725142 - National Science Foundation; Beckman Foundation; Sherman Fairchild Foundation; John Stauffer Charitable Trust; Christian Scholars Foundation)Published versionSupporting documentatio

A high-throughput, whole cell assay to identify compounds active against carbapenem-resistant Klebsiella pneumoniae.

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

Homodimericin A: A Complex Hexacyclic Fungal Metabolite

Microbes sense and respond to their environment with small molecules, and discovering these molecules and identifying their functions informs chemistry, biology, and medicine. As part of a study of molecular exchanges between termite-associated actinobacteria and pathogenic fungi, we uncovered a remarkable fungal metabolite, homodimericin A, which is strongly upregulated by the bacterial metabolite bafilomycin C1. Homodimericin A is a hexacyclic polyketide with a carbon backbone containing eight contiguous stereogenic carbons in a C20 hexacyclic core. Only half of its carbon atoms have an attached hydrogen, which presented a significant challenge for NMR-based structural analysis. In spite of its microbial production and rich stereochemistry, homodimericin A occurs naturally as a racemic mixture. A plausible nonenzymatic reaction cascade leading from two identical achiral monomers to homodimericin A is presented, and homodimericin A’s formation by this path, a six-electron oxidation, could be a response to oxidative stress triggered by bafilomycin C1

Small Molecule DFPM Derivative-Activated Plant Resistance Protein Signaling in Roots Is Unaffected by EDS1 Subcellular Targeting Signal and Chemical Genetic Isolation of victr R-Protein Mutants

The small molecule DFPM ([5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione) was recently shown to trigger signal transduction via early effector-triggered immunity signaling genes including EDS1 and PAD4 in Arabidopsis thaliana accession Col-0. Chemical genetic analyses of A. thaliana natural variants identified the plant Resistance protein-like Toll/Interleukin1 Receptor (TIR)-Nucleotide Binding (NB)-Leucine-Rich Repeat (LRR) protein VICTR as required for DFPM-mediated root growth arrest. Here a chemical genetic screen for mutants which disrupt DFPM-mediated root growth arrest in the Col-0 accession identified new mutant alleles of the TIR-NB-LRR gene VICTR. One allele, victr-6, carries a Gly216-to-Asp mutation in the Walker A domain supporting an important function of the VICTR nucleotide binding domain in DFPM responses consistent with VICTR acting as a canonical Resistance protein. The essential nucleo-cytoplasmic regulator of TIR-NB-LRR-mediated effector-triggered immunity, EDS1, was reported to have both nuclear and cytoplasmic actions in pathogen resistance. DFPM was used to investigate the requirements for subcellular EDS1 localization in DFPM-mediated root growth arrest. EDS1-YFP fusions engineered to localize mainly in the cytoplasm or the nucleus by tagging with a nuclear export signal (NES) or a nuclear localization signal (NLS), respectively, were tested. We found that wild-type EDS1-YFP and both the NES and NLS-tagged EDS1 variants were induced by DFPM treatments and fully complemented eds1 mutant plants in root responses to DFPM, suggesting that enrichment of EDS1 in either compartment could confer DFPM-mediated root growth arrest. We further found that a light and O2-dependent modification of DFPM is necessary to mediate DFPM signaling in roots. Chemical analyses including Liquid Chromatography-Mass Spectrometry and High-Resolution Atmospheric Pressure Chemical Ionization Mass Spectrometry identified a DFPM modification product that is likely responsible for bioactivity mediating root growth arrest. We propose a chemical structure of this product and a possible reaction mechanism for DFPM modification

Growth arrest of lateral roots when Arabidopsis roots were exposed to fresh DFPM for a prolonged period.

<p>A) Transfer of wild-type Col-0 plants to DFPM medium one time inhibited primary root growth, while lateral roots could elongate further (arrowheads). Images were taken 7 days after the first transfer. DFPM-insensitive <i>victr-1</i> was used as a control. B) Prolonged exposure to fresh DFPM by re-transferring seedlings daily for 4 days resulted in further inhibition of growth in lateral roots and leaves in wild type (Col-0). Wild type (Col-0) and <i>victr-1</i> seedlings were transferred to 10 μM DFPM or DMSO control medium one time (A) or every 24 hours for 4 days (B). The black horizontal bars indicate the primary root lengths at the time of first transfer to DFPM. Primary and lateral roots of <i>victr-1</i> grew normally in all cases similarly to non-treated controls. C) Lengths of Col-0 and <i>victr-1</i> lateral roots in B were measured and shown as frequency distribution. n = 2, 12–19 plants per experiment. D) Lateral roots of Col-0 plants in (B) were swollen and bent similar to typical symptoms for primary roots treated with DFPM [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0155937#pone.0155937.ref015" target="_blank">15</a>]. Scale bar applies for both images.</p

DFPM-induced root growth arrest is unaffected by the subcellular targeting signals of EDS1-YFP protein.

<p>A) EDS1-YFP localization in the root of EDS1-YFP, EDS1-YFP-NES and EDS1-YFP-NLS in <i>eds1-2</i>. All transgenic EDS1-YFP constructs were driven by the native EDS1 promoter. Interestingly, EDS1-YFP signals increased after 24 hours of 10 μM DFPM application compared to the non-treated controls. Scale bar applies to all 6 images. Confocal gain and pinhole parameters were identical in all six images. At least 3 plants were observed for DFPM treated condition. B-C) Both NES- or NLS-tagged EDS1 protein versions expressed in <i>eds1-2</i> were capable of complementing the <i>eds1-2</i> phenotype in DFPM-mediated primary root growth arrest. Seven day-old seedlings were exposed to 10 μM DFPM. Three days after treatment, two independent lines of EDS1-YFP-NES and EDS1-YFP-NLS showed the same DFPM sensitivity as wild-type Col-0 and EDS1-YFP control (B, C). A mutated control EDS1-YFP-nes also showed a similar sensitivity to DFPM. Means with different letters are grouped based on two-way ANOVA and Tukey’s test, <i>P</i> < 0.05). Error bars represent SD (n = 8 to 12 seedlings per condition for 10 μM DFPM, n = 2 to 4 for 0 μM DFPM). Representative plants are shown in (C). The black horizontal bars mark the root tip position when plants were exposed to DFPM. The vertical black bars separate the indicated genotypes grown on the same plate for clarity.</p

LC-MS analyses of DFPM modification in the presence or absence of light.

<p>DFPM in aqueous solutions gave a distinct absorption peak in non-treated control sample (t = 0) (λ_max = 318 nm, blue arrowheads). A) This peak declined after 180 and 360 min DFPM solution incubation in the light. At the same time a second peak was detected which accumulated over time in the light (grey arrowhead). B) If the same solution was incubated in the dark, the DFPM peak continued to decline but more slowly over time (blue arrowheads). No secondary metabolite was detected in the absence of light (B).</p