TLC analysis of C12-3-oxo-AHL produced by parent strain WCS358 (lane 1), by mutant derivative IBE4 (lane 2) and mutant derivative carrying a plasmid expressing Lon IBE4 (pBBRlon) (lane 3)

<p><b>Copyright information:</b></p><p>Taken from "The Lon protease is involved in -acyl homoserine lactone quorum sensing regulation"</p><p>http://www.biomedcentral.com/1471-2180/7/71</p><p>BMC Microbiology 2007;7():71-71.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1949823.</p><p></p> Lane 4 contains the standards C10-3-oxo-AHL and C12-3-oxo-AHL. The AHLs were visualized with AHL-sensor strain (pSB1075); a volume corresponding to 5 × 10cfu was loaded on the TLC assay

Western hybridization performed using anti-PpuR antibody on total cellular protein extracts

<p><b>Copyright information:</b></p><p>Taken from "The Lon protease is involved in -acyl homoserine lactone quorum sensing regulation"</p><p>http://www.biomedcentral.com/1471-2180/7/71</p><p>BMC Microbiology 2007;7():71-71.</p><p>Published online 26 Jul 2007</p><p>PMCID:PMC1949823.</p><p></p> Total protein extracts were performed using different protein amounts of either parent strain WCS358 or mutant derivative IBE4 each carrying a plasmid expressing PpuR (pBBRppuR). Lane 1 corresponds to 10 μg of purified PpuR-6His; lane 2 and lane 3 correspond to total proteins from 2 × 10cfu of WCS358(pBBRppuR) and IBE4(pBBRppuR) respectively; lanes 4 and 5 correspond to total proteins from 10cfu. For lanes 4 and 5 the images were scanned using a Versadoc (Biorad) and the QuantityOne software; results of this quantification are displayed as a histogram. See text for all details

Schematic representation of QteE- and RsaL-dependent regulation of the <i>P. aeruginosa las</i> QS system.

<p>In the pre-quorum period, QteE binds to the LasR receptor and prevents the binding of the LasR-3OC₁₂-HSL complex to the <i>rsaL</i>-<i>lasI</i> bidirectional promoter <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112105#pone.0112105-Siehnel1" target="_blank">[20]</a>, hence delaying the onset of the QS response. Once the quorum has been reached, the LasR/3OC₁₂-HSL complex triggers the transcription of both <i>rsaL</i> and <i>lasI</i> genes. The consequent increase of 3OC₁₂-HSL levels, and thus of activated LasR, generates a positive feedback loop also responsible for the increase of RsaL levels. RsaL binding to the <i>rsaL</i>-<i>lasI</i> bidirectional promoter represses the expression of both <i>rsaL</i> and <i>lasI</i> genes, thus counteracting the positive feedback loop. This circuit provides 3OC₁₂-HSL homeostasis <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112105#pone.0112105-Rampioni3" target="_blank">[24]</a>. Solid arrows represent positive control; T-shaped lines represent negative control; dashed arrows indicate information flow; curved arrows represent the transcription start points of the indicated genes.</p

Effect of QS dysregulation caused by <i>qteE</i> mutation on pyocyanin production in <i>P. aeruginosa</i>.

<p>Levels of pyocyanin measured in cell-free supernatants from cultures of the indicated strains grown till A₆₀₀≈3.5. C, no plasmid; EV, pBBR1MCS-5 empty vector; pQteE, pBBR1MCS-5 derivative plasmid for <i>qteE</i> expression.</p

Effect of QS dysregulation caused by <i>qteE</i> mutation on <i>P. aeruginosa</i> virulence-related phenotypes.

<p>Levels of (A) 3OC₁₂-HSL, (B) elastase, (C) proteases produced along growth by <i>P. aeruginosa</i> wild type (black lines), <i>qteE</i> (red lines) and <i>rsaL</i> strains (green line) carrying the pBBR1MCS-5 empty vector, or by the <i>qteE</i> strain carrying the pQteE plasmid (pBBR1MCS-5-derived) for the expression of <i>qteE</i> (dashed red line). Values are the means (± standard deviations) of at least three independent experiments.</p

Effect of QS dysregulation caused by <i>qteE</i> and <i>rsaL</i> mutations on <i>P. aeruginosa</i> pathogenesis in mice.

<p>C57Bl/6 mice were infected with the indicated strains embedded in agar beads. (A) Mice mortality induced by bacteremia (light grey) and survival (white) were evaluated on challenged mice. (B) Clearance (white) and capacity to establish chronic airways infection (dark grey) were determined on surviving mice after 14 days from challenge. The results are averages of three independent experiments. Statistical significance is indicated by an asterisk comparing <i>P. aeruginosa</i> wild type versus <i>qteE</i> or <i>rsaL</i> strains (<i>p</i><0.05).</p

Effect of the induction of P<i>lasR</i>-bound proteins on P<i>lasR</i> promoter activity.

<p>(A) Histogram reporting P<i>lasR</i> maximal promoter activity (grey bars) and the corresponding cell density (white bars) measured in <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> strains carrying the plasmids indicated below the graph, grown in LB supplemented with 0.1% (w/v) l-arabinose. (B) Graph reporting P<i>lasR</i> promoter activity (filled symbols) and cell density (open symbols) measured during the growth curve in <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> carrying pHERD30T (triangles) or pR3699 (circles), grown in LB supplemented with 0.1% (w/v) l-arabinose. (C) Histogram reporting P<i>lasR</i> maximal promoter activity measured in <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> strains carrying pHERD30T (grey bars) or pR3699 (white bars) grown in LB supplemented with different l-arabinose concentrations (%, w/v), indicated below the graph. In (A), (B) and (C) the average of three independent experiments is reported with standard deviations; in (A) and (C) statistical significance with respect to <i>P. aeruginosa</i> PAO1 P<i>lasR::lux</i> (pHERD30T) is indicated with one asterisk (<i>p</i> < 0.01).</p

P<i>lasR</i> activity and P<i>lasR</i>-affinity chromatography.

<p>(A) Growth curve of the <i>P. aeruginosa</i> PAO1 wild type strain carrying the pMP<i>lasR</i>::<i>lacZ</i> plasmid (dashed line) and corresponding P<i>lasR</i> promoter activity (solid line). The points of the growth curve at which the protein crude extracts for DNA-affinity chromatography were prepared are indicated by arrows. (B) SDS-PAGE analysis of proteins bound to the P<i>lasR</i> promoter region. Protein crude extracts were prepared from <i>P. aeruginosa</i> PAO1 cultures grown in LB broth to the indicated cell densities (A₆₀₀). Bands analysed by MALDI-TOF mass spectrometry are indicated by arrows, and the corresponding protein name is reported.</p

Effect of PA3699 induction on <i>P. aeruginosa</i> virulence factors production.

<p>Histogram reporting elastase, pyocyanin and proteases production measured in <i>P. aeruginosa</i> PAO1 carrying pHERD30T (grey bars) or pR3699 (white bars), grown in LB supplemented with 0.1% (w/v) l-arabinose. The average of three independent experiments is reported with standard deviations; statistical significance with respect to <i>P</i>. <i>aeruginosa</i> PAO1 (pHERD30T) is indicated with one asterisk (<i>p</i> < 0.01).</p

PA3699 purification and P<i>lasR</i>-binding assay.

<p>(A) SDS-PAGE analysis of samples withdrawn at different steps of PA3699 purification. Lane L, PageRuler Unstained Protein Ladder (Fermentas); lane 1, non-induced protein crude extract; lane 2, induced protein crude extract; lane 3, soluble fraction of the induced protein crude extract; lane 4, purified protein; line 5, purified protein after thrombin cleavage. (B) Western blot analysis performed with mouse anti-6xHis primary antibody and anti-mouse peroxidase-conjugated secondary antibody on a gel identical to the one shown in (A). (C) Autoradiography of an EMSA showing direct interaction between a DNA probe encompassing the <i>lasR</i> promoter region and purified PA3699. PA3699 concentration (μM) is indicated above each lane. An unspecific probe was added in the reaction mixture as control. The PA3699-P<i>lasR</i> complex and the free DNA probes are indicated.</p