The absence of SigX modulated <i>P</i>. <i>aeruginosa</i> virulence in the <i>C</i>. <i>elegans</i> model.

<p>Kaplan-Meier survival plots of <i>C</i>. <i>elegans</i> nematodes fed with the wild type strain <i>P</i>. <i>aeruginosa</i> H103 (n = 210), the <i>sigX</i> mutant PAOSX (n = 257), and the SigX-complemented mutant strain PAOSX+ (n = 201). Each value reported for the assay is the mean of measurements of eight samples from three independent experiments. Pairwise strain comparisons (log rank test) were as follows: H103 <i>versus</i> PAOSX, <i>p</i>-value< 0.0001; PAOSX <i>versus</i> PAOSX+, <i>p</i>-value< 0.0001; H103 <i>versus</i> PAOSX+, <i>p</i>-value <0.001. Four independent experiments were performed. </p

Altered levels of (A) exotoxinA, (B) pyocyanin and (C) siderophores in the culture supernatants of the <i>sigX</i> mutant.

<p>H103 (black), PAOSX (white) and PAOSX+ (grey) culture supernatants were obtained from overnight cultures in (A) LB, (B) King A or (C) King B media. The relative amounts of exotoxin A, pyocyanin and the total siderophores were assayed at least three times independently for each strain and means and standard deviations are presented. For the Chrome Azurol S (CAS) assay, the haloes around the wells in the CAS plate show siderophore production in sample supernatant. Statistics were done by pairwise strain comparisons (<i>t</i> test). *<i>p</i>-value<0.05, **<i>p</i>-value<0.01, ***<i>p</i>-value<0.001, **** <i>p</i>-value<0.0001, NS no significant difference.</p

Functional classes of SigX-regulated genes identified by expression profiling on DNA array.

<p>All 307 genes that had a significant difference in expression between wildtype and mutant strains (Fold change ≥2,<i>p</i>-value ≤0.05 as determined by Empirical Bayes) were included and classified according to their function. Functional classes were determined using the <i>Pseudomonas</i> Genome Project website (<a href="http://www.pseudomonas.com" target="_blank">www.pseudomonas.com</a>; Winsor et al., 2011), among which these framed in grey were discussed.</p

Involvement of SigX in (A) twitching and (B) swarming motilities, attachment to (C) glass slides and to (D) Caco2/TC7 cells, and (E) biofilm formation.

<p>Twitching motility was assayed on solidified M9G medium containing 1% agar. Swarming motility was assayed on M9G containing 1% casamino acids as nitrogen source and solidified with 0.5 % agar. For adherence onto glass slides, mid-log phase cultures of GFP expressing bacteria were diluted in 0.9% NaCl to an OD₅₈₀ of 0.6 and allowed to adhere for 2h. Attached cells were observed using a confocal laser scanning microscope and a binding index was calculated (value on each slide). Binding of bacteria onto Caco2/TC7 cells: each bar represents the mean number of adherent bacteria per cell (±SD) calculated by direct microscopic counting of 100 cells and expressed as a percentage compared to the binding of the wildtype H103 strain. For biofilm assay, bacteria were allowed to form a pellicle for 24h at 37°C. Biofilms were quantified by measuring the absorbance at 595 nm after crystal violet (CV) staining. Relative biofilm formation was determined by comparison to the wildtype strain (±SD). Each experiment was performed at least three times. Statistics were done by pairwise strain comparisons (<i>t</i> test). *<i>p</i>-value<0.05, **<i>p</i>-value<0.01, ***<i>p</i>-value<0.001.</p