Nitric oxide signaling in Pseudomonas aeruginosa biofilms mediates phosphodiesterase activity, decreased cyclic di-GMP levels, and enhanced dispersal

Abstract

Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.

FIG. 1.

NO-mediated dispersal in P. aeruginosa involves PDE activity and decreased levels of intracellular c-di-GMP. (A) Induction of P. aeruginosa biofilm dispersal by the addition of the NO donor SNP in continuous cultures. Five-day-old biofilms were exposed to 5 μM SNP, and the induction of dispersal was measured by comparing CFU counts released in the effluent runoff with those for the untreated control biofilms. (B) Addition of GTP inhibits the degradation of the PDE-specific substrate bis-pNPP by intact P. aeruginosa cells in a dose-dependent manner. PDE activity was quantified as p-nitrophenol production at OD₄₀₅. (C) Inhibition of NO-mediated dispersal in P. aeruginosa by GTP. Preestablished P. aeruginosa biofilms were treated with (white bars) or without (dark bars) SNP at 500 nM as previously described (5) in the absence or presence of 250 μM or 1 mM GTP; then, the biofilms on the slides were quantified (percent surface coverage) using digital image analysis. (D) Low doses of NO stimulate PDE activity in cell extracts of P. aeruginosa. Cell extracts of P. aeruginosa were incubated with 5 mM bis-pNPP in the presence of various concentrations of SNP and in the presence (white bars) or absence (dark bars) of 100 μM of the NO scavenger PTIO. The release of p-nitrophenol was quantified at OD₄₀₅ after 2 h. (E) Effect of low concentrations of NO on c-di-GMP levels in P. aeruginosa. (i) Five-day-old P. aeruginosa wild-type and ΔnirS mutant biofilms were treated with 5 μM SNP for 1 h to initiate dispersal or were left untreated before nucleotides were extracted from biofilm cells as described in Materials and Methods. (ii) P. aeruginosa in suspension cultures was exposed to various concentrations of SNP for 2 h, and nucleotides were extracted. c-di-GMP was detected in the nucleotide samples, quantified by LC-MS-MS, analysis and referred to in mg of cellular protein. Data are mean values, and error bars indicate standard errors of the means (n = 3).

FIG. 2.

Low doses of NO induce physiological changes in P. aeruginosa. (A) Pyoverdine production of P. aeruginosa cells was quantified by fluorescence measurement (excitation, 355 nm; emission, 460 nm) after growth in King's B medium in the presence or absence of low doses of SNP. (B) Expression of the denitrification gene nirS. A P. aeruginosa reporter strain, NSGFP, which expresses GFP under the control of the nirS promoter, was used to monitor the relative expression levels of the denitrification gene nirS in planktonic cells exposed to various concentrations of SNP. Data are mean values, and error bars indicate standard errors of the means (n = 3).

FIG. 3.

NO-mediated dispersal in P. aeruginosa knockout mutants of selected genes. The mutants were assessed in a batch culture biofilm dispersal assay in the presence or absence of 500 nM SNP; then, the biofilms on the slides were stained with SYTO 9 to allow analysis using fluorescence microscopy and were quantified (percent surface coverage) using digital image analysis. (A) The images show microscopic pictures of the biofilms on glass slides treated without (−) or with (+) 500 nM SNP. Bar, 50 μm. (B) The bars show levels of biofilm surface coverage when treated with 500 nM SNP (white bars) or untreated controls (dark bars). Data are mean values, and error bars indicate standard errors of the means (n ≥ 3). ns, not significant.

FIG. 4.

(A) Biofilms of the BdlA mutant strain, KO[bdlA], do not disperse when exposed to low doses of NO in continuous-flow cultures. Five-day-old biofilms of the P. aeruginosa wild-type and KO[bdlA] mutant strains were exposed to 5 μM SNP for 1 h. Subsequently, the dispersal of cells was determined by CFU counts in the biofilm effluent runoff and compared to the level for an untreated control. (B) Effect of low concentrations of NO on c-di-GMP levels in the P. aeruginosa wild-type and KO[bdlA] strains. P. aeruginosa wild-type (dark bars) and KO[bdlA] mutant (white bars) cells in suspension cultures were exposed to 5 μM and 50 μM SNP and controls for 2 h, and c-di-GMP was extracted and quantified by using LC-MS-MS. Data are mean values, and error bars indicate standard errors of the means (n = 3).