Article Figures & Data
Figures
- FIG. 1.
The soxR deletion does not compromise P. aeruginosa's fitness in standard in vitro culture conditions. Growth curves for P. aeruginosa wt carrying vector pME6001 (squares), P. aeruginosa ΔsoxR (circles), and P. aeruginosa ΔsoxR/pSoxR (triangles). The experiment was conducted in a 96-multiwell plate platform. Plates loaded with tryptic soy broth supplemented with gentamicin (50 μg ml−1) and inoculated with P. aeruginosa strains (OD600 of 0.1) were incubated (37°C, 18 h) in a SpectraMax Plus Microplate Reader (Molecular Devices) and OD600 was measured every 60 min with 10 s of shaking before each reading. The data represent means of triplicates ± standard errors.
- FIG. 2.
P. aeruginosa (Pa) SoxR activates expression of six P. aeruginosa genes in response to PQ treatment. Scatter plots of mean gene intensity values comparing (A) PQ-treated P. aeruginosa wt and P. aeruginosa ΔsoxR strains, (B) untreated P. aeruginosa wt and ΔsoxR strains, and (C) H2O2-treated P. aeruginosa wt and P. aeruginosa ΔsoxR strains. The lines above and below the diagonal indicate the intensity thresholds that correspond to a twofold increase (inner lines) and a fivefold decrease (outer lines) in mRNA levels, respectively. Plots show the means of gene intensity from six arrays. Only the genes represented by the data points inside the oval in plot A showed statistically significant expression changes after statistical treatment of the data sets from plots A, B, and C with the Wilcoxon-Mann-Whitney test.
- FIG. 3.
E. coli and S. enterica serovar Typhimurium SoxR binding site-like sequences are found in the promoters of transcriptional units regulated by P. aeruginosa SoxR. (A) Gene loci with P. aeruginosa SoxR-regulated genes. The gene loci are depicted as reported in the annotated P. aeruginosa genome (http://www.pseudomonas.com/current_annotation.asp ). The genes of the proposed P. aeruginosa SoxR regulon are in boldface. The open arrows mark the positions of P. aeruginosa SoxR-regulated promoters. (B) Alignment of E. coli (1) and S. enterica serovar Typhimurium (2) SoxR binding sites found in the promoters of the respective soxS genes and the predicted P. aeruginosa soxR binding sites found upstream of PA2274 (3), the mexGHI-ompD operon (4), and PA3718 (5). The conserved SoxR binding sites are in boldface inside the box. The middle of the dyad symmetrical sequence is marked with a dot. The E. coli soxS transcriptional start site is marked +1. The −10 and −35 boxes of the E. coli soxS promoter and the predicted −10 and −35 boxes in the other promoters are indicated. The arrow marks the start codon of the SoxR proteins in sequences 1, 2, and 3. The number of nucleotides to the start codons of SoxS, PA2274, MexG, and PA3718 is indicated.
- FIG. 4.
Deletion of soxR does not reduce the ability of P. aeruginosa to withstand peroxide and superoxide-based oxidative stress. (A) PQ susceptibility assay. (B) H2O2 susceptibility assay. Paper disks impregnated with PQ and H2O2 solutions were placed onto agar plates seeded with P. aeruginosa wt/pME6001 (circles), P. aeruginosa ΔsoxR/pME6001 (squares), and P. aeruginosa ΔsoxR/pSoxR (triangles) to obtain homogeneous bacterial lawns, and the zones of growth inhibition around the disks were recorded after incubation (24 h, 37°C). In the plot, a zone of growth inhibition of 6 mm corresponds to the diameter of the disk and indicates that there was no detectable inhibition zone. The data represent means of triplicates ± standard errors.
- FIG. 5.
P. aeruginosa SoxR is required for virulence in a mouse pulmonary infection model. The plot shows the results of a survival experiment following a pulmonary challenge with P. aeruginosa in a mouse model. Mice were infected with a lethal dose (106 CFU) of agarose-encapsulated P. aeruginosa wt cells (squares) or an equivalent dose of P. aeruginosa ΔsoxR (circles) or P. aeruginosa ΔsoxR/pSoxR (triangles).
Tables
- TABLE 1.
Genes whose mRNA levels increased exclusively in PQ-treated cells and in a soxR-dependent manner
Gene no./name P. aeruginosa wt treated with: P. aeruginosa ΔsoxR treated with: P. aeruginosa ΔsoxR/pSoxR treated with PQ (fold change by RT-PCR) PQ H2O2 PQ H2O2 Fold change by arraya Fold change by RT-PCRb Fold change by array Fold change by RT-PCR Fold change by array Fold change by RT-PCR Fold change by array Fold change by RT-PCR PA2274 20.7 ± 3.4 35.9 ± 0.3 1.4 ± 0.2 0.9 ± 0.3 1.0 ± 0.1 1.3 ± 0.5 1.2 ± 0.2 2.8 ± 0.3 78.0 ± 0.2 PA3718 3.4 ± 0.2 6.5 ± 0.1 1.1 ± 0.1 1.0 ± 0.4 0.6 ± 0.1 1.8 ± 0.3 1.1 ± 0.4 0.7 ± 0.4 73.5 ± 0.8 PA4205/mexG 16.5 ± 2.6 7.1 ± 0.5 1.4 ± 0.3 0.4 ± 0.3 1.1 ± 0.2 0.4 ± 0.5 1.1 ± 0.4 2.1 ± 0.5 41.2 ± 0.8 PA4206/mexH 12.3 ± 1.6 14.2 ± 0.4 1.5 ± 0.2 0.9 ± 0.3 1.3 ± 0.3 1.6 ± 0.5 1.0 ± 0.2 1.7 ± 0.2 31.3 ± 0.2 PA4207/mexI 9.4 ± 1.4 5.8 ± 0.3 1.5 ± 0.2 0.9 ± 0.3 0.9 ± 0.1 0.7 ± 0.4 1.0 ± 0.2 2.2 ± 0.3 13.0 ± 0.3 PA4208/opmD 9.0 ± 1.4 5.9 ± 0.6 1.3 ± 0.1 1.3 ± 0.7 0.9 ± 0.1 1.2 ± 1.5 1.0 ± 0.2 1.4 ± 1.5 8.9 ± 2.5 ↵ a Fold change values were calculated as the ratio of the means of gene signals of PQ- or H2O2-treated and untreated cultures. Fold change values are shown with standard errors. Each mean was derived from six arrays.
↵ b Fold changes values were calculated as described in Materials and Methods. Values represent means of triplicates ± standard errors.
