The phosphodiesterase DipA (PA5017) is essential for Pseudomonas aeruginosa biofilm dispersion

Abstract

Although little is known regarding the mechanism of biofilm dispersion, it is becoming clear that this process coincides with alteration of cyclic di-GMP (c-di-GMP) levels. Here, we demonstrate that dispersion by Pseudomonas aeruginosa in response to sudden changes in nutrient concentrations resulted in increased phosphodiesterase activity and reduction of c-di-GMP levels compared to biofilm and planktonic cells. By screening mutants inactivated in genes encoding EAL domains for nutrient-induced dispersion, we identified in addition to the previously reported ΔrbdA mutant a second mutant, the ΔdipA strain (PA5017 [dispersion-induced phosphodiesterase A]), to be dispersion deficient in response to glutamate, nitric oxide, ammonium chloride, and mercury chloride. Using biochemical and in vivo studies, we show that DipA associates with the membrane and exhibits phosphodiesterase activity but no detectable diguanylate cyclase activity. Consistent with these data, a ΔdipA mutant exhibited reduced swarming motility, increased initial attachment, and polysaccharide production but only somewhat increased biofilm formation and c-di-GMP levels. DipA harbors an N-terminal GAF (cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA) domain and two EAL motifs within or near the C-terminal EAL domain. Mutational analyses of the two EAL motifs of DipA suggest that both are important for the observed phosphodiesterase activity and dispersion, while the GAF domain modulated DipA function both in vivo and in vitro without being required for phosphodiesterase activity. Dispersion was found to require protein synthesis and resulted in increased dipA expression and reduction of c-di-GMP levels. We propose a role of DipA in enabling dispersion in P. aeruginosa biofilms.

Fig 1

Modulation of c-di-GMP levels and phosphodiesterase activity over the course of biofilm development by P. aeruginosa wild-type and dipA mutant strains. c-di-GMP levels (A) and specific phosphodiesterase activity (B) were determined using cell extracts obtained from P. aeruginosa PA14 planktonic, dispersed, and biofilm cells and biofilms remaining attached following dispersion (“biofilms post dispersion”). c-di-GMP/mg refers to c-di-GMP levels per total cell pellet protein (in mg). *, significantly different from biofilms (P < 0.05), as determined by ANOVA and SigmaStat. Experiments were repeated at least 5 times. Similarly, specific phosphodiesterase activity (B) was determined using cell extracts obtained from ΔdipA mutants grown planktonically and as a biofilm prior to and following addition of glutamate. **, significantly different based on Student's t test (P < 0.01). Experiments were repeated at least 4 times. (C) Domain overview of DipA and RbdA. GAF, cGMP-specific phosphodiesterases, adenylyl cyclases, and FhlA domain; PAS, Per Arnt Sim sensory domain; EAL, phosphodiesterase domain; GGDEF, diguanylate cyclase domain; aa, amino acid.

Fig 2

DipA and RbdA are essential for the dispersion response of P. aeruginosa PA14. (A) ΔdipA and ΔrbdA biofilms do not disperse in response to addition of glutamate, ammonium chloride, nitric oxide, and mercury chloride compared to wild-type P. aeruginosa PA14. Biofilms were grown in tube reactors. (B) Confocal images of ΔbifA and ΔdipA biofilms prior to and 30 min following addition of glutamate. Biofilms were grown in flow cells for 5 days before induction of dispersion. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Invitrogen Corp.). White size bars = 100 μm.

Fig 3

DipA plays a minor role in biofilm formation by P. aeruginosa. (A) Biofilms of strains PA14 and PAO1 inactivated in or (over)expressing dipA, grown for 144 h, were visualized by CLSM. P. aeruginosa biofilms in the absence/presence of the empty plasmid (pMJT1) were used as controls. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Invitrogen Corp.). White size bars = 100 μm. (B) c-di-GMP levels of P. aeruginosa PA14 biofilms inactivated in or (over)expressing dipA. “c-di-GMP/mg” refers to c-di-GMP levels per total cell pellet protein (in mg) used. *, significantly different from PA14 biofilms (P < 0.05), as determined by ANOVA and SigmaStat. (C) The dispersion-deficient phenotype of ΔdipA biofilms is restored upon complementation with dipA in trans. Biofilms were grown for 5 days before dispersion was induced by a sudden increase in the medium glutamate concentration. Biofilm effluents were collected for a period of 18 min, and the absorbance was determined at 600 nm. Determination of c-di-GMP levels was repeated at least 6 times, while all other experiments were carried out in triplicate. Error bars indicate standard deviations.

Fig 4

Dispersion by wild-type P. aeruginosa biofilms correlates with increased expression of dipA and requires protein synthesis. (A) Fold change in dipA, rbdA, and bdlA RNA levels in wild-type P. aeruginosa planktonic cells, the remaining biofilms following dispersion, and dispersed cells compared to biofilm growth conditions was assessed by qRT-PCR analysis. (B) P. aeruginosa biofilms following 4 days of growth under flowing conditions were treated for 30 min with tetracycline (arrow 1) prior to the induction of dispersion (arrow 2). Control biofilms were not treated. Dispersion was induced by a sudden increase in the medium glutamate concentration. The biofilm effluent was collected in 0.2-ml aliquots into microtiter plates, and the absorbance was read at 600 nm. All experiments were carried out in triplicate. Error bars indicate standard deviations. *, significantly different from biofilms (P < 0.05), as determined by ANOVA and SigmaStat.

Fig 5

Inactivation of dipA impairs swarming motility but enhances initial attachment and polysaccharide levels at the posttranscriptional level. (A) Swarming motility of P. aeruginosa PA14 strains inactivated or (over)expressing dipA following 48 h of growth on M8 agar. (B) Swarming motility of P. aeruginosa PAO1 strains inactivated or (over)expressing dipA, dipA-NoGAF, GAFonly, or dipA harboring an alanine substitution in either one of the EAL motifs. The swarming diameter was recorded following 24 h of growth on M8 agar. Insets above the graph are representative images showing swarming motility of selected strains. (C) Initial attachment of the P. aeruginosa PAO1, ΔdipA, and PAO1/pMJT-dipA strains following 6 h of growth using the microtiter plate dish assay and crystal violet staining. (D) Psl polysaccharide levels relative to the PAO1 parental strain as determined using anti-Psl dot blot analysis. (E) pelA and pslA are not differentially expressed in PAO1 and PA14 strains inactivated in or overexpressing dipA compared to the parental strains, as determined qRT-PCR analysis. *, significantly different from PAO1 (P < 0.05), as determined by ANOVA and SigmaStat.

Fig 6

dipA encodes a membrane-associated phosphodiesterase. (A) Immunoblot analysis of subcellular cell fractions (15 μg) demonstrating that V5/6×His-tagged DipA is located in the cytoplasm and associated with the membrane. Subcellular localization was achieved by ultracentrifugation. (B) V5/6×His-tagged DipA purified in the presence of the detergent Tween 20 is partly soluble, as indicated by a portion of the protein precipitating after ultracentrifugation and not remaining detectable in the supernatant. A total of 28 μg of purified DipA and 10 μg of the resulting supernatant fraction (Sup) and precipitate (pellet) were loaded onto the SDS gel. (C) DipA is a phosphodiesterase, as determined using purified V5/6×His-tagged DipA and bis(p-nitrophenyl) phosphate as a substrate. c-di-GMP concentrations ranging from 4 to 100 pmol were added to the enzyme assay. cAMP and cGMP were added at a final concentration of 100 μM. *, significantly different from DipA (P < 0.05). (D) Degradation of c-di-GMP by purified DipA and DipA-NoGAF (100 μg each) over a period of 240 min, as determined by HPLC analysis. cAMP was added at a final concentration of 1 μM. *, significantly different from DipA (P < 0.05). (E) Effect of overexpression of intact and truncated dipA and mutated dipA on aggregative behavior of PAO1/pJN-PA4843, as indicated by turbidity of liquid growth culture. *, significantly different from PAO1 harboring empty vector (P < 0.05). Aggregative behavior was assessed by turbidity. Following 3 h of growth under planktonic conditions at 37°C in LB, arabinose was added to a final concentration of 1%. Following 3.5 h of continued incubation, the bacterial suspensions were allowed to settle at room temperature for 10 min before the absorbance of the suspension was measured at 600 nm. Cultures not induced by arabinose were used as controls. (F) Effect of overexpression of intact and truncated dipA and mutated dipA on swarming motility of PAO1/pJN-PA4843 following 24 h of growth on M8 agar. *, significantly different from PAO1/pJN105 (P < 0.05). All experiments were carried out at least 5 times. Error bars indicate standard deviations. C, cytoplasmic fraction; M, membrane fraction; sup, supernatant after ultracentrifugation; pellet, pellet after ultracentrifugation; TCE, total cell extract; purified, DipA was purified using Ni-NTA affinity chromatography; No DipA, PAO1/pMJT1 cell extract not expressing any His-tagged DipA was purified using Ni-NTA affinity chromatography, and the respective eluates were used as controls; N.D., not detected. Significance was determined by ANOVA and SigmaStat.

Fig 7

The EAL motifs are essential for dispersion, while the GAF domain is not essential for dispersion and biofilm formation. (A) Both EAL motifs are essential for dispersion. (B) Dispersion assays demonstrating that complementation of the ΔdipA mutant with dipA-NoGAF but not GAFonly restored the biofilm-deficient phenotype of the ΔdipA mutant to wild-type levels. (C) Biofilm architecture of biofilms expressing dipA-NoGAF. Biofilms were grown for 6 days in diluted LB medium, after which time, confocal images were acquired. Biofilms were stained with the LIVE/DEAD BacLight viability stain (Invitrogen Corp.). White size bars = 100 μm. All experiments were carried out at least in triplicate. Error bars indicate standard deviations.