Inhibition of <i>Arabidopsis</i> hypocotyl extension by hexylresorcinol.

<p>All plants were grown for 5 days in the dark. A. Average length of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol. Error bars represent the standard deviation of the samples. B. Percentage germination of seedlings grown on 20, 30, 40 50, 80 or 100 µM hexylresorcinol.</p

Growth of <i>M. smegmatis</i> mc²155 in the presence of mitoxantrone or suramin.

<p>A. Bacteria were grown in liquid cultures in the presence of drug for 22 hours, and then plated onto drug-free agar. After a further 48 h the colonies were counted for each concentration of drug. B. Colony counts for bacteria grown in the presence of 0, 13 or 65 µM mitoxantrone for 16, 19 or 22 hours before being plated onto solid media.</p

<i>In vitro</i> characterisation of DNA gyrase screen hits.

<p>A. Determination of the IC₅₀ for mitoxantrone in a supercoiling assay with 1 unit of gyrase (12 nM); 100 μM ciprofloxacin (Cip.) was used as a positive control for inhibition. The positions of relaxed (Rel.) and negatively supercoiled (SC) DNA are indicated. B. Determination of the IC₅₀ for suramin. C. Assaying the abilities of mitoxantrone and suramin to induce gyrase-mediated DNA cleavage. The reactions were carried out in the absence of ATP. Ciprofloxacin was used as a positive control for gyrase-mediated cleavage. The position of linear DNA (Lin.) is indicated. D. Suramin-induced protection of DNA from Ca²⁺-induced, gyrase-mediated cleavage. E. Inhibition of gyrase binding to a 147 bp DNA fragment by suramin.</p

<i>In vitro</i> characterisation of topoisomerase VI screen hits.

<p>A. Determination of IC₅₀ values for hits in the presence of 1 unit (50 nM) of <i>M. mazei</i> topo VI. This resulted in the following IC₅₀ values: 6 μM for 9-aminoacridne; 30 μM for m-amsacrine; 30 μM for suramin; 30 μM for hexylresorcinol; 40 μM for purpurin; 8 μM for quinacrine; and 2 μM for mitoxanthrone. B. Native gel shift assays for the binding of <i>M. mazei</i> topo VI to a 147 bp DNA fragment in the presence of screen hits.</p

Summary of Screen Hits.

*<p>Not determined in this study.</p

DNA cleavage assays with topoisomerase VI screen hits.

<p>A. Assaying the abilities of screen hits to induce <i>M. mazei</i> topo VI-mediated DNA cleavage with 1 unit topo VI (50 nM). B. Inhibition of <i>S. shibatae</i> topo VI by screen hits. C. Assaying the abilities of screen hits to induce <i>S. shibatae</i> topo VI-mediated DNA cleavage. D. Protection of DNA from ADPNP-induced, <i>S. shibatae</i> topo VI-mediated cleavage by screen hits.</p

Percentage of amino acid sequence identity between <i>Agl97</i>, <i>Agl1</i>, <i>Agl2</i>, <i>Agl3</i> and <i>Agl4</i>.

<p>Genbank accession numbers are shown.</p

Time course of development of maltase and α-glucosidase activity in the endosperm.

<p>Extracts were prepared from endosperm pooled from five seedlings at each time point. Values are means of three technical replicates ± SE. Circles, without inhibitor; triangles, with 0.5 mM DNJ in the assay. Upper graph, assay with pNPG as the substrate. Lower graph, assay with maltose as the substrate.</p

Biomolecular Characterization of the Levansucrase of Erwinia amylovora, a Promising Biocatalyst for the Synthesis of Fructooligosaccharides

Erwinia amylovora is a plant pathogen that affects Rosaceae, such as apple and pear. In <i>E. amylovora</i> the fructans, produced by the action of a levansucrase (EaLsc), play a role in virulence and biofilm formation. Fructans are bioactive compounds, displaying health-promoting properties in their own right. Their use as food and feed supplements is increasing. In this study, we investigated the biomolecular properties of EaLsc using HPAEC-PAD, MALDI-TOF MS, and spectrophotometric assays. The enzyme, which was heterologously expressed in Escherichia coli in high yield, was shown to produce mainly fructooligosaccharides (FOSs) with a degree of polymerization between 3 and 6. The kinetic properties of EaLsc were similar to those of other phylogenetically related Gram-negative bacteria, but the good yield of FOSs, the product spectrum, and the straightforward production of the enzyme suggest that EaLsc is an interesting biocatalyst for future studies aimed at producing tailor-made fructans

Fractionation on a MonoQ anion exchange column of maltase activity from endosperm.

<p>All fractionations were reproduced at least twice, on independent batches of seedlings. Representative examples are shown. (A), (B). Extracts of endosperm of seedlings of cv NFC Tipple at 10 dpi were applied to a MonoQ column by FPLC. Proteins binding the column were eluted with a gradient of increasing NaCl concentration (dashed line). One-mL fractions were collected and assayed for α-glucosidase activity with pNPG (A), or maltase activity with maltose (B). I-III indicate the three peaks of activity. (C). Extracts were prepared, fractionated and assayed for maltase as in (B), except that seedlings were either transgenic lines in a cv Golden Promise background carrying an RNA interference (RNAi) silencing cassette for <i>Agl97</i> [lines 21 and 23 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0151642#pone.0151642.ref031" target="_blank">31</a>] (squares)], or an out-segregant line (i.e. not carrying the silencing cassette) from the same transformation experiment (circles). Chromatographic conditions were the same as in (B) except that the column wash to remove unbound proteins was reduced from 20 to 10 mL. Note that peak II maltase activity elutes at the same NaCl concentration in the experiments shown in (B) and (C). (D), (E). Fractionation on a MonoS column of proteins not binding to a MonoQ column [peak I in (A) and (B)]. Proteins binding to the MonoS column were eluted with a gradient of increasing NaCl concentration (dashed line). One-mL fractions were collected and assayed for α-glucosidase activity with pNPG (D), or for maltase activity with maltose (E). Peaks of activity eluting from the column are designated Ia-Id.</p