Discovery of (Dihydro)pyrazine <i>N</i>‑Oxides via Genome Mining in Pseudomonas

Overexpression of the Pseudomonas <i>virulence factor</i> (<i>pvf</i>) biosynthetic operon led to the identification of a family of pyrazine <i>N-</i>oxides (PNOs), including a novel dihydropyrazine <i>N,N</i>′-dioxide (dPNO) metabolite. The nonribosomal peptide synthetase responsible for production of (d)­PNOs was characterized, and a biosynthetic pathway for (d)­PNOs was proposed. This work highlights the unique chemistry catalyzed by <i>pvf</i>-encoded enzymes and sets the stage for bioactivity studies of the metabolites produced by the virulence pathway

Lipopeptides from the Tropical Marine Cyanobacterium <i>Symploca</i> sp.

A collection of the tropical marine cyanobacterium <i>Symploca</i> sp., collected near Kimbe Bay, Papua New Guinea, previously yielded several new metabolites including kimbeamides A–C, kimbelactone A, and tasihalide C. Investigations into a more polar cytotoxic fraction yielded three new lipopeptides, tasiamides C–E (<b>1</b>–<b>3</b>). The planar structures were deduced by 2D NMR spectroscopy and tandem mass spectrometry, and their absolute configurations were determined by a combination of Marfey’s and chiral-phase GC-MS analysis. These new metabolites are similar to several previously isolated compounds, including tasiamide (<b>4</b>), grassystatins (<b>5</b>, <b>6</b>), and symplocin A, all of which were isolated from similar filamentous marine cyanobacteria

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

Design, Synthesis, and Antifungal Activity of 3‑Substituted-2(<i>5H</i>)‑Oxaboroles

Next generation antimicrobial therapeutics are desperately needed as new pathogens with multiple resistance mechanisms continually emerge. Two oxaboroles, tavaborole and crisaborole, were recently approved as topical treatments for onychomycosis and atopic dermatitis, respectively, warranting further studies into this privileged structural class. Herein, we report the antimicrobial properties of 3-substituted-2(5H)-oxaboroles, an unstudied family of medicinally relevant oxaboroles. Our results revealed minimum inhibitory concentrations as low as 6.25 and 5.20 μg/mL against fungal (e.g., Penicillium chrysogenum) and yeast (Saccharomyces cerevisiae) pathogens, respectively. These oxaboroles were nonhemolytic and nontoxic to rat myoblast cells (H9c2). Structure–activity relationship studies suggest that planarity is important for antimicrobial activity, possibly due to the effects of extended conjugation between the oxaborole and benzene rings

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

Mapping of new <i>victr</i> mutant alleles isolated via facile DFPM-mediated root growth arrest.

<p>A) Overview and amino acid changes (marked with stars) in three newly isolated EMS mutants in the <i>VICTR</i> locus. B) EMS point mutants in <i>VICTR</i> (Col-0), <i>victr-6</i>, <i>victr-7 and victr-8</i>, showed insensitivity to DFPM similarly to T-DNA mutant <i>victr-1</i> whereas the wild-type Col-0 control was fully arrested in root growth assays. The black horizontal bars mark the root tip position when plants were exposed to 10 μM DFPM. The vertical grey bars separate the indicated genotypes grown on the same plate for clarity. C) Quantitative analyses of root growth assays shown in B). With 10 μM DFPM all three EMS <i>victr</i> point mutants and <i>victr-1</i> showed a similar DFPM insensitivity compared to the their growth in 0 μM DFPM. Wild type Col-0 showed a strong inhibition in root growth upon DFPM treatment. Error bars represent SEM (n = 7–18 seedlings for 10 μM DFPM, n = 3–17 for 0 μM DFPM).</p

Natural Product Screen and Follow-up.

<p>(A) Scatter plot of the normalized luminescence scores for the two original replicates of the natural product screening data from <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162686#pone.0162686.s005" target="_blank">S1 Table</a> (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0162686#pone.0162686.s006" target="_blank">S2 Table</a> for data from the plates retested with and without antimycin). The data are plotted on a Log2 scale. The extract plates included DMSO wells and empty wells, which are plotted as the “negative controls.” The positive control plates contained wells with 2.4 mM idebenone, which was diluted upon transfer to the cell plate. These points are labeled “idebenone.” (B) Seahorse data for the three compounds isolated from <i>Chimaphila umbellata</i>. (C) Seahorse data for the four compounds isolated from <i>Sterospermum euphoroides</i>. For (B) and (C), each experiment was performed three times and data from a single representative experiment is shown.</p

A Maldiisotopic Approach to Discover Natural Products: Cryptomaldamide, a Hybrid Tripeptide from the Marine Cyanobacterium <i>Moorea producens</i>

Genome sequencing of microorganisms has revealed a greatly increased capacity for natural products biosynthesis than was previously recognized from compound isolation efforts alone. Hence, new methods are needed for the discovery and description of this hidden secondary metabolite potential. Here we show that provision of heavy nitrogen ¹⁵N-nitrate to marine cyanobacterial cultures followed by single-filament MALDI analysis over a period of days was highly effective in identifying a new natural product with an exceptionally high nitrogen content. The compound, named cryptomaldamide, was subsequently isolated using MS to guide the purification process, and its structure determined by 2D NMR and other spectroscopic and chromatographic methods. Bioinformatic analysis of the draft genome sequence identified a 28.7 kB gene cluster that putatively encodes for cryptomaldamide biosynthesis. Notably, an amidinotransferase is proposed to initiate the biosynthetic process by transferring an amidino group from arginine to serine to produce the first residue to be incorporated by the hybrid NRPS-PKS pathway. The maldiisotopic approach presented here is thus demonstrated to provide an orthogonal method by which to discover novel chemical diversity from Nature

Validation of Complex I Deficient Cell Line and Bypass Screening Assay.

<p>(A) Western blot of wild-type and <i>Ndufa9</i> knockout C2C12 cells. (B) Complex I and complex IV dipstick assays for wild-type and <i>Ndufa9</i> knockout C2C12 cells, with technical triplicates shown for each. (C) Seahorse comparing wild-type and <i>Ndufa9</i> knockout C2C12 cells. (D) Seahorse of <i>Ndufa9</i> knockout cells (left) and rotenone pre-treated wild-type C2C12s (right), each treated with a dose-response of idebenone. Doses are indicated in the legend below each plot. (E) Luminescence-based complex I bypass assay utilizing <i>Ndufa9</i> knockout cells. Cells treated with a dose-response of idebenone in the presence or absence of 125 nM antimycin. For (A-D), each experiment was performed three times, and a single representative experiment is shown. For (E) the data shown represent the average +/- SEM of data from three independent experiments.</p