Phylogenetic Relationships of Taxa Related to FI-1004.

<p>The evolutionary history was inferred using the Neighbor-Joining method. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Saitou1" target="_blank">[50]</a> The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1000 replicates) are shown next to the branches. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura2" target="_blank">[51]</a> and are in the units of the number of base substitutions per site. The analysis involved 24 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1353 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura1" target="_blank">[30]</a> The sequence of <i>Brevibacterium linens</i> DSM 20425^T was used as an outgroup.</p

Fish Origins and Taxonomy, Microbial Isolates, and NCBI Closest Relatives.

<p>Fish Origins and Taxonomy, Microbial Isolates, and NCBI Closest Relatives.</p

Bioactivities of Fish Microbiome Isolates.

<p>A = Actinobacteria, F = Firmicutes, α = Alphaproteobacteria, γ = Gammaproteobacteria, (−) = Gram-negative, (+) = Gram-positive. Check mark indicates activity in growth inhibition assay.</p

Chemical Structure for Sebastenoic Acid.

<p>a, b and c are subunits found using 2D NMR methods. HMBC correlations depicted by solid arrows, COSY correlations depicted by bold lines, NOESY correlations depicted by dashed arrows.</p

Molecular Phylogenetic Analysis by Maximum Likelihood for all Isolated, Culturable Strains of Bacteria Isolated from Fish Intestines.

<p>The evolutionary history was inferred by using the Maximum Likelihood method based on the Kimura 2-parameter model. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Kimura1" target="_blank">[48]</a> The bootstrap consensus tree inferred from 2000 replicates is taken to represent the evolutionary history of the taxa analyzed. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Branches corresponding to partitions reproduced in less than 50% bootstrap replicates are collapsed. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (2000 replicates) are shown next to the branches. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Felsenstein1" target="_blank">[49]</a> Initial tree(s) for the heuristic search were obtained automatically as follows. When the number of common sites was <100 or less than one fourth of the total number of sites, the maximum parsimony method was used; otherwise BIONJ method with MCL distance matrix was used. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+<i>G</i>, parameter = 0.4869)). The rate variation model allowed for some sites to be evolutionarily invariable ([+<i>I</i>], 38.9340% sites). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. The analysis involved 53 nucleotide sequences. All positions containing gaps and missing data were eliminated. There were a total of 1244 positions in the final dataset. Evolutionary analyses were conducted in MEGA5. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035398#pone.0035398-Tamura1" target="_blank">[30]</a> Strains identified as psychrophilic bacteria in NCBI denoted with blue diamonds. Strains whose closest published NCBI relatives are uncultured clones denoted with open triangles.</p

Sebastenoic Acid (1) MICs Against Bacterial Panel.

<p>Sebastenoic Acid (1) MICs Against Bacterial Panel.</p

Development of an activity-based probe for acyl-protein thioesterases

<div><p>Protein palmitoylation is a dynamic post-translational modification (PTM) important for cellular functions such as protein stability, trafficking, localization, and protein-protein interactions. S-palmitoylation occurs via the addition of palmitate to cysteine residues via a thioester linkage, catalyzed by palmitoyl acyl transferases (PATs), with removal of the palmitate catalyzed by acyl protein thioesterases (APTs) and palmitoyl-protein thioesterases (PPTs). Tools that target the regulators of palmitoylation–PATs, APTs and PPTs–will improve understanding of this essential PTM. Here, we describe the synthesis and application of a cell-permeable activity-based probe (ABP) that targets APTs in intact mammalian cells and the parasite <i>Toxoplasma gondii</i>. Using a focused library of substituted chloroisocoumarins, we identified a probe scaffold with nanomolar affinity for human APTs (HsAPT1 and HsAPT2) and synthesized a fluorescent ABP, JCP174-BODIPY TMR (JCP174-BT). We use JCP174-BT to profile HsAPT activity <i>in situ</i> in mammalian cells, to detect an APT in <i>T</i>. <i>gondii</i> (TgPPT1). We show discordance between HsAPT activity levels and total protein concentration in some cell lines, indicating that total protein levels may not be representative of APT activity in complex systems, highlighting the utility of this probe.</p></div

JCP174-BT labels HsAPT1, HsAPT2 and TgPPT1 in intact cells.

<p>A, Intact U-2 OS mammalian cells were labeled with different concentrations of JCP174-BT, washed to remove unbound probe, and lysed. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate doublet of interest ~25 kDa previously identified as HsAPT1 and HsAPT2. B, Intact U-2 OS mammalian cells were labeled with JCP174-BT, washed to remove unbound probe, and lysed. HsAPT1 was immunoprecipitated from the lysate, with input, supernatant, and elution samples resolved by SDS-PAGE. Fluorescent probe signal was visualized via flatbed scanner (upper panel). HsAPT1 identity was confirmed by western blot (lower panel). The red caret indicates HsAPT1 species on the fluorescent scan. C, Intact <i>T</i>. <i>gondii</i> tachyzoites were labeled with different concentrations of JCP174-BT, washed to remove unbound probe and lysed. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate bands of interest ~31 kDa. D, Intact wild-type <i>T</i>. <i>gondii</i> or ΔTgPPT1 tachyzoites were labeled with JCP174-BT as in (C). Carets indicate species corresponding to TgPPT1 in labeling of wild-type tachyzoites.</p

JCP174-BT labels HsAPT1, HsAPT2 and TgPPT1 <i>in vitro</i>.

<p>A, Structure of JCP174-BODIPY TMR (JCP174-BT) composed of chloroisocoumarin small-molecule JCP174 (red) and BODIPY TMR fluorophore (yellow). B, C, In-gel ABP competition labeling with JCP174-BT. (B) Purified recombinant HsAPT1 or HsAPT2 was pre-treated with different concentrations of JCP174, palmostatin B, or DMSO (0) before labeling with JCP174-BT. Samples were resolved by SDS-PAGE and fluorescent probe signal visualized using a flatbed scanner (upper panel), with loading assessed via silver stain (lower panel). (C) Purified recombinant TgPPT1 WT and active site mutant (S128A) were treated with JCP174, palmostatin B or DMSO (0) and labeled with JCP174-BT. Samples were analyzed as in (B).</p

JCP174-BT profiles APT activity in intact mammalian cells and <i>T</i>. <i>gondii</i> parasites.

<p>A, Intact wild-type <i>T</i>. <i>gondii</i> or ΔTgPPT1 tachyzoites were labeled with JCP174-BT or FP-rho, washed to remove unbound probe and lysed. <i>T</i>. <i>gondii</i> tachyzoite lysate was labeled with FP-rho. Lysates were resolved by SDS-PAGE with fluorescent probe labeling visualized using a flatbed scanner. Carets indicate species corresponding to TgPPT1 in labeling of wild-type tachyzoites. Asterisk (*) indicates m-Cherry expressed in ΔTgPPT1 parasites. B, Intact U-2 OS cells were labeled with JCP174-BT or FP-rho, washed to remove unbound probe, and lysed. U-2 OS lysate was labeled with FP-rho. Lysates were resolved via SDS-PAGE. Fluorescent signal was visualized with a flatbed scanner (fluorescence scan), with the coomassie stain of the same gel shown to indicate loading. Carets indicate the species corresponding to HsAPT1 and HsAPT2. C, Intact mammalian cells were labeled with JCP174-BT and processed as in (A). Pairs of oncogenic cell lines from derived from three tissue types (carcinomas of the breast, ovary and prostate) were chosen to contrast low metastatic/aggressive potential (MCF7, OVCAR-3, and LNCaP) versus high metastatic potential (MDA-MB-231, SKOV-3, and PC-3) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0190255#pone.0190255.ref025" target="_blank">25</a>]. For each oncogenic pair, wedges indicate low to high metastatic potential. Fluorescent signal was visualized with a flatbed scanner (top panel). Total HsAPT1 protein level was visualized by western blot (middle panel). GAPDH was used as a loading control (lower panel).</p