<i>motif-x</i> analyses for PKA (A and B) and CK II (C and D).

<p>These motif extraction results illustrate the inter-residue correlations found among the phosphorylated peptides identified using the ProPeL methodology, and are highly consistent with the previously established consensus sequences for the PKA and CK II kinases.</p

Receiver Operating Characteristic (ROC) curves for the <i>scan-x</i> and Scansite PKA and CK II kinase specific predictors.

<p>These curves illustrate the tradeoff between sensitivity and specificity achieved by the ProPeL based <i>scan-x</i> (red) and combinatorial peptide library based Scansite (blue) predictors, and indicate the similarity of results achieved using these experimentally orthogonal approaches. Panels (A) and (B) are based on PKA serine and threonine predictions, respectively, while panels (C) and (D) are based on CK II serine and threonine predictions, respectively. The Scansite web server does not score all phosphorylatable residues in a given sequence, which results in partial ROC curves.</p

Top 20 <i>scan-x</i> CK II phosphorylation predictions based on a human whole proteome scan with the CK II motif obtained using the ProPeL methodology.

*<p>Out of 1,168,144 total serine and threonine residues.</p>**<p>From the PhosphoSitePlus database.</p>***<p>Tryptic peptide containing the predicted phosphorylation site greater than 35 residues in length.</p>****<p>Phosphorylated at homologous site in rat and cow.</p

Comparison of combinatorial peptide library screening, proteome-derived library, and ProPeL motif discovery methodologies.

*<p>Phosphorylatable residues (Ser and Thr) and Cys are not included in combinatorial peptide libraries used for kinase specificity determination.</p>**<p>In proteome-derived libraries motif width limits depend on whether the kinase reaction is performed before or after proteolytic peptide digestion.</p

Isolation and identification of three novel natural compounds.

<p>Chromatogram obtained during fractionation of the crude extract CR1223-D showing three peaks corresponding to the three compounds isolated. Compound 1 was further identified as 6-propyl gentisyl alcohol, compound 2 as 5-hydroxy-4-(hydroxymethyl)-2-methyl 2,3-dihydrobenzofuran, and compound 3 as 2-(hydroxymethyl)-3-propyl benzoquinone.</p

Spectrophotometric assay for bacterial sugar fermentation.

<p>Absorbance spectrum of MM^Suc alone (indicator) or incubated with wild-type <i>V. cholerae</i> (WT) or a PTS mutant for 5 hours. The spectra are shown at the left, while the visible color difference is shown in microtiter dish wells at the right. The largest difference in absorbance between MM^Suc incubated with wild-type <i>V. cholerae</i> and that incubated with a PTS mutant is measured at 615 nm (red arrow).</p

Impact of compounds 1, 2, and 3 on <i>V. cholerae</i> sugar fermentation and growth.

<p>The assays were carried out at 30°C in pH-MM^Suc to monitor sugar fermentation by A₆₁₅ (A, B, and C) or in MM^Pyr to monitor bacterial growth by OD₆₁₅ (D, E, and F). Bacteria were exposed to mirandamycin (A and D), compound 2 (B and E) or compound 3 (C and F) at concentrations ranging from 14 to 383 µM. Replicate assay is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031307#pone.0031307.s002" target="_blank">Figure S2</a>.</p

Minimum inhibitory concentrations (MICs) of mirandamycin and known antibiotics against selected bacterial pathogens.

(*)<p>MIC was determined by Alamar Blue Assay as described in Material and Methods; Mirandamycin (MIR), levofloxacin (LEVO), ampicillin (AMP), imipenem (IMI), bactrim (TMP/SMX), isoniazid (INH), pyrazinamide (PZA), ethambutol: ETH.</p

Representative results for secondary screen.

<p>Bacteria were grown in MM^Pyr, pH-MM^Suc, or pH-MM^Glu. OD₆₁₅ measurements of cultures in MM^Pyr reflect the ability of cells to grow in the presence of extract, while absorbance measurements in pH-MM^Suc and pH-MM^Glu reflect the ability of cells to transport and ferment these sugars in the presence of extract. Data are shown for wild-type <i>V. cholerae</i> and a PTS mutant in the absence of extract (A,B) or for wild-type <i>V. cholere</i> in the presence of extracts that we hypothesize (C) interfere with sugar transport and fermentation, (D) inhibit bacterial growth (bacteriostatic), or (E) kill bacteria (bactericidal).</p

Flow chart of HTS assay.

<p>The HTS assay begins with manual preparation of working solutions of pH-MM^Suc and a bacterial suspension with OD₆₀₀ of 0.015 in PBS. Subsequent steps of the assay are fully automated: solution mixing in the 384 well-plates is performed by a ThermoScientific Matrix WellMate liquid dispenser, pin-transfer of the natural extracts tested is done with a custom-built Epson robot, and A₆₁₅ readout after incubation at room temperature is accomplished at 6 and 20 hours using an EnVision™ multi-well spectrophotometer. Finally, EnVision™ data were analyzed with Spotfire™ and Excel. Each assay is performed in duplicate. A measurement was considered to be statistically significant if it deviated by at least three standard deviations from the mean measurement calculated using all measurements made with a particular extract library. The calculated Z′ factor for the screen was 0.808±0.088.</p