Environmental regulation of Pseudomonas aeruginosa PAO1 Las and Rhl quorum-sensing systems

Abstract

The lasI-lasR and the rhlI-rhlR quorum-sensing systems in Pseudomonas aeruginosa regulate the expression of numerous cellular and secreted virulence factor genes and play important roles in the development of biofilms. The las and rhl systems themselves are known to be directly or indirectly regulated by a number of transcriptional regulators, and consequently, their expression is sensitive to environmental conditions. In this report, the activities of these two quorum-sensing systems have been examined systematically under 46 growth conditions, and the regulation by environmental conditions has been investigated. The relative timing and strength of expression of these two systems varied significantly under different conditions, which contrasts with the notion of a preset hierarchy with these two systems in P. aeruginosa. Depending on the growth conditions, the correlation between each synthase and its cognate transcriptional regulator also varied, suggesting that the transcription of these genes independently allows for further fine tuning of each system. Finally, we observe that the activities of both the lasI-lasR and the rhlI-rhlR quorum-sensing systems were dramatically enhanced in the presence of extracts of sputum samples from cystic fibrosis patients.

FIG. 1.

Correlation between synthase gene expression and HSL production. (A) lasI expression profile in TSB-DC-EDDA medium and (B) 3-oxo-C12-HSL production as measured by the A. tumefaciens detection system. (C) rhlI expression profile in M9 medium and (D) C4-HSL production as measured by the PAO-JP2 (pKD-rhlA) system. Autoinducer production is presented as relative HSL units, i.e., the cps value minus the control cps value divided by the OD value.

FIG. 2.

Cluster of lasI-lasR and rhlI-rhlR temporal expression profiles under 40 growth conditions. To compare the relative expression levels of each gene across all conditions, the data were normalized by the maximum values under all 40 conditions for each gene. The profiles were grouped by hierarchical clustering using average linkage analysis performed with the Cluster program and visualized using Treeview (7).

FIG. 3.

Comparison of expression profiles of the lasI, lasR, rhlI, rhlR, aprA, and rhlA promoters under two growth conditions, LB and 1/4-diluted LB. The expression profiles of the promoter-reporter fusions were measured every 30 min for 30 h at 37°C. Only every second data point is shown. Panels A to F represent the profiles of lasI, lasR, aprA, rhlI, rhlR, and rhlA, correspondingly. Solid diamonds and open triangles represent expression in LB and 1/4-diluted LB, respectively. The data are presented as cps × 10⁻³ (kcps) and are the average values for four repeats, with the standard deviations indicated by the vertical bars.

FIG. 4.

Distribution of cell densities at which maximum QS gene expression was observed under 42 growth conditions. Shown are the OD values at which the maximal expression of each gene for each condition was plotted. The 40 conditions presented in Table 1 as well as growth in the presence of CF sputum extracts at two concentrations (Fig. 6) are presented. Solid diamonds, open diamonds, solid circles, and open circles represent data for lasI, lasR, rhlI, and rhlR, respectively.

FIG. 5.

Correlations of expression levels of synthase and regulator genes. The maximal expression values for the synthase genes (lasI and rhlI) were plotted against the maximal expression values of the regulator genes (lasR and rhlR) for all 40 conditions as for Fig. 2. R² values are calculated from the linear regression of the data.

FIG. 6.

Activation of QS gene expression by sputum extracts. The expression profiles of the promoter-reporter fusions were measured every 30 min for 30 h at 37°C in M9 minimal medium with glucose. The data are presented as cps × 10⁻³ (kcps) normalized to OD₆₂₀. Panels A to F represent the profiles of lasI, lasR, aprA, rhlI, rhlR, and rhlA, correspondingly. Diamonds, triangles, and circles represent no addition, 2.5% sputum extract, and 5% sputum extract, respectively. Only every second data point is shown.

FIG. 7.

Expression profiles of rhlI, rhlR, and rhlA under microaerophilic conditions and the effect of NaCl. The expression profiles of the promoter-reporter fusions were measured every 3 h. The cultures were placed in an anaerobic jar and incubated at 37°C. (A) rhlI; (B) rhlR; (C) rhlA. Diamonds and open triangles represent expression with no addition and addition of 100 mM NaCl, respectively. The data are presented as cps × 10⁻³ (kcps) and are not normalized. Growth curves are represented by dashed lines.