Figure 1.
PlasR activity and PlasR-affinity chromatography.
(A) Growth curve of the P. aeruginosa PAO1 wild type strain carrying the pMPlasR::lacZ plasmid (dashed line) and corresponding PlasR 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 PlasR promoter region. Protein crude extracts were prepared from P. aeruginosa 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.
Figure 2.
Effect of the induction of PlasR-bound proteins on PlasR promoter activity.
(A) Histogram reporting PlasR maximal promoter activity (grey bars) and the corresponding cell density (white bars) measured in P. aeruginosa PAO1 PlasR::lux strains carrying the plasmids indicated below the graph, grown in LB supplemented with 0.1% (w/v) l-arabinose. (B) Graph reporting PlasR promoter activity (filled symbols) and cell density (open symbols) measured during the growth curve in P. aeruginosa PAO1 PlasR::lux carrying pHERD30T (triangles) or pR3699 (circles), grown in LB supplemented with 0.1% (w/v) l-arabinose. (C) Histogram reporting PlasR maximal promoter activity measured in P. aeruginosa PAO1 PlasR::lux 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 P. aeruginosa PAO1 PlasR::lux (pHERD30T) is indicated with one asterisk (p < 0.01).
Figure 3.
PA3699 purification and PlasR-binding assay.
(A) SDS-PAGE analysis of samples withdrawn at different steps of PA3699 purification.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 lasR 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-PlasR complex and the free DNA probes are indicated.
Figure 4.
Effect of PA3699 induction on P. aeruginosa virulence factors production.
Histogram reporting elastase, pyocyanin and proteases production measured in P. aeruginosa 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 P. aeruginosa PAO1 (pHERD30T) is indicated with one asterisk (p < 0.01).