Inhibition of acyl-HSL synthases by substrate analogs.

<p>The best-fit models of inhibition are graphed. The µM concentration of inhibitor for each experiment is shown next to the curve. A) Substrate-velocity curves of mixed inhibition of 0.4 µM BmaI1 by octyl-ACP. B) Substrate-velocity curves of competitive inhibition of 0.5 µM BjaI with varying isopentyl-CoA.</p

Substrates and products of acyl-HSL synthases.

<p>A) Acyl-HSL synthases have two substrates and three products. The substrate acyl group is attached as a thioester to an acyl carrier: either an acyl carrier protein or coenzyme A<b>.</b> B) Comparison of the structures of acyl-ACP and acyl-CoA. Both carriers have an acyl-phosphopantetheine (acyl-PPant) moiety. Thioether analogs of these thioester substrates lack the acyl oxygen.</p

Kinetics of inhibition by sulfide analogs.

<p>Kinetics of inhibition by sulfide analogs.</p

Kinetic constants for members of the acyl-HSL synthase family.

a<p>RhlI kinetic constants are from another study <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#pone.0112464-Raychaudhuri1" target="_blank">[33]</a>.</p>b<p><i>k</i>_cat/<i>K</i>_m ratio = (<i>k</i>_cat/<i>K</i>_m)^{preferred substrate}/(<i>k</i>_cat/<i>K</i>_m)^{non-preferred substrate}.</p><p>Kinetic constants for members of the acyl-HSL synthase family.</p

Sources of Diversity in Bactobolin Biosynthesis by <i>Burkholderia thailandensis</i> E264

A series of deletion mutants in the recently identified bactobolin biosynthetic pathway defined the roles of several key biosynthetic enzymes and showed how promiscuity in three enzyme systems allows this cluster to produce multiple products. Studies on the deletion mutants also led to four new bactobolin analogs that provide additional structure–activity relationships for this interesting antibiotic family

Structures of the acyl-substrate recognition motif.

<p>A) Alignment of the crystal structures of LasI (1R05 in blue) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#pone.0112464-Watson1" target="_blank">[18]</a> and EsaI (1KZF in red) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#pone.0112464-Gould1" target="_blank">[19]</a>. The two structures have a root-mean-square deviation of 1.45 Å for 124 amino acid α carbons. The conserved α-helix proposed to interact with ACP is circled in yellow. The active site cleft is behind this helix next to the conserved β-sheet. B) The LasI structure rotated 90° about the Z axis with positively-charged residues in the motif displayed.</p

Chemoenzymatic synthesis of octyl-ACP sulfide.

<p>A) Synthesis of octyl ACP. In this two-step reaction, octyl-CoA sulfide was first synthesized by coupling octyl bromide with Coenzyme A, followed by enzymatic transfer of the alkyl-PPant to apo-ACP using <i>Bacillus subtilis</i> Sfp PPant transferase (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112464#s4" target="_blank">materials and methods</a>). B) Mass spectrum of purified octyl-ACP. The intensity is relative to the largest peak of 8960 Da. The expected mass is 8957 Da.</p

Identification of new LasR binding sites.

<p>(A) Location of LasR boxes of genes relative to the TSSs. Most LasR binding sites (LasR box) are localized at 51–52 nt upstream to the TSS of the genes. The binding site is shown as a line upstream to the TSS (blue, red, and purple; novel binding sites, previously known binding sites, and genes with multiple binding sites, respectively). (B) Positioning of the LasR box (blue line) is immediately upstream to the sigma factor binding site (green line). Black arrow represents the TSS. (C) EMSA of DNA fragments from predicted LasR-regulated genes. The concentrations of purified LasR used in each assay are shown above the reactions. DNA fragments from the promoter region of the <i>rsaL</i> and an internal fragment from the <i>hrpA</i> gene were used as positive and negative controls, respectively.</p

Characterization of the LasR-regulated small RNAs Lrs1 and Lrs2.

<p>(A) Growth (lines) and expression of the <i>lrs1</i> or <i>lrs2</i> reporter constructs (bars) in wild-type or Δ<i>lasR P. aeruginosa</i> PA14 are shown. (B) Role of Hfq in controlling the levels of Lrs1 and Lrs2 sRNAs. Gel shift analyses of sRNAs interaction with increasing amounts of purified Hfq are shown. Lane 5 in both panels contains, in addition to labeled probe, a 100-fold excess of unlabeled Lrs1 or Prrf1 RNA. (C) Lower panels show RT-PCR analyses of Lrs1 and Lrs2 expression in <i>P. aeruginosa</i> wild-type and Δ<i>hfq</i>. Prrf1 and RsmZ were used as positive and negative controls, respectively, for sRNAs interacting and not interacting with Hfq.</p

Distribution of temperature regulated genes, and segments transcribing antisense RNAs and intergenic small RNAs in the genome of <i>P. aeruginosa</i> PA14.

<p>The outer circle represents the core (blue) and accessory genes (yellow) as defined previously <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002945#ppat.1002945-Mathee1" target="_blank">[9]</a>. The innermost two circles represent genes that were differentially expressed at 28°C and 37°C (blue and orange, upregulated in 28°C and 37°C, respectively); the middle two circles show the expression of sRNAs from intergenic regions (red dots) and asRNAs (green dots). For each category, relative ranking of expression was grouped into five color-coded bins (darker colors represent higher expression).</p