Modulation of Pseudomonas aeruginosa biofilm dispersal by a cyclic-Di-GMP phosphodiesterase with a putative hypoxia-sensing domain

Abstract

Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.

FIG. 1.

The biofilm phenotypes of P. aeruginosa PAO1 and its rbdA transposon insertion mutants. (A) Genetic organization and domain structures of RbdA. Vertical arrows indicate the relative transposon insertion sites in each mutant. The domain structure prediction was performed using the SMART program (http://smart.emblheidelberg.de/). Abbreviations: SP, signal peptide; TM, transmembrane domain; PAS, PAS domain; PAC, PAC domain; GGDEF, diguanylate cyclase domain; EAL, diguanylate phosphodiesterase domain. (B) Visualization of bacterial biofilm formation on polystyrene tubes by crystal violet staining. (C) Quantitative comparison of the biofilm formation by wild-type strain PAO1 and its rbdA mutants. The data shown are means of triplicates, and the standard deviations are shown by error bars.

FIG. 2.

RbdA is associated with biofilm dispersal. (A) Time course analysis of bacterial biofilm mass. (B) In trans expression of rbdA from the multicopy vector pUCP19 in PAO1 and the ΔrbdA deletion mutant reduced biofilm accumulation. The empty vector pUCP19 was introduced into the wild type and ΔrbdA mutant, and these constructs were used as controls. (C) Expression of a single copy of rbdA in the ΔrbdA mutant restored the biofilm phenotype to the level of wild-type PAO1. The integration vector Tn7 was inserted in PAO1 and the ΔrbdA mutant, and these constructs were used as controls. The data shown are means of triplicates, and the standard deviations are shown by error bars.

FIG. 3.

RbdA is a c-di-GMP phosphodiesterase. (A) The standard control mixture containing four nucleotides in a final concentration of 50 μm was prepared in the reaction buffer, and 10 μl was injected for HPLC analysis. (B to D) RbdA after reaction with c-di-GMP at room temperature for 10 min (B), at room temperature for 10 min followed by 37°C for 30 min (C), and at room temperature for 10 min followed by 37°C for 240 min (D). (E and F) RbdA after reaction with GTP at 37°C for 0 min (E) or 120 min (F). The peak areas (μV·s) of key molecules are provided under the corresponding HPLC peak for the convenience of comparison. (G) RbdA and the phosphodiesterase BifA, but not the diguanylate cyclase SadC, were functional homologues in regulation of biofilm development.

FIG. 4.

GTP induction of RbdA phosphodiesterase activity requires the GGDEF domain. (A) The phosphodiesterase activity of RbdA and its variant GGDEF-5A in the absence and presence of GTP. The concentration of c-di-GMP after termination of the reaction was determined by measuring UV absorbance following HPLC separation. The experiment was performed twice with similar results, and the figure shows a representative set of data. (B) In vivo assay of the role of the GGDEF domain in modulation of biofilm development. The ΔrbdA deletion mutant was complemented by in trans expression of the wild-type rbdA or its variant, GGDEF-5A, from the vector pUCP19. The inset shows the Western blot data for RbdA and its variant, GGDEF-5A, expressed in the ΔrbdA mutant.

FIG. 5.

The PAS domain is critical for RbdA activity. (A) The PAS domain of RbdA shares the conserved structural folds and the key residues of the PAS domain of FixL (NCBI accession no. CAA40143). The α-helix and β-sheet of FixL (dark shading) are based on previous reports (17, 23), and the secondary structure of RbdA was predicted using the program PredictProtein (http://cubic.bioc.columbia.edu/). The identical, highly similar, and similar residues are indicated by the symbols *, :, and ., respectively. The arrows indicate the key amino acid residues of FixL involved in heme and oxygen binding (30). (B) Quantification of biofilm formation of strain PAO1 and its derivatives under aerobic conditions. The ΔrbdA deletion mutant was complemented by in trans expression of the wild type rbdA or its variants. The inset shows the Western blot data for RbdA and its variants expressed in the ΔrbdA mutant performed with protein samples prepared from bacterial cultures when the OD₆₀₀ reached about 1.5.

FIG. 6.

Bacterial growth and biofilm development of strain PAO1 and its derivatives under aerobic and anaerobic conditions. (A) Time course analysis of bacterial growth. For Western blot analysis (inset), total protein samples were prepared from anaerobically grown bacterial cultures when the OD₆₀₀ reached about 1.5. (B) Time course analysis of biofilm formation.

FIG. 7.

RbdA regulates bacterial motility, EPS production, and pellicle formation. (A) Swarming motility; (B) swimming motility; (C) rhamnolipid production; (D) EPS production; (E) pellicle formation in static culture (top right) and after crystal violet staining (bottom right). Three plates were used for each swarming and swimming test, and data shown are the averages of three independent experiments and the standard deviations.

FIG. 8.

RbdA modulates biofilm formation through its influence on the genes encoding EPS biosynthesis. (A) Congo red-binding analysis of EPS production by PAO1 and its derivatives. (B) The effects of pel and psl operon deletions on pellicle formation. (C) pelA′-lacZ fusion gene expression assay. Different bacterial strains containing the PpelA-lacZ construct were grown in LB broth at 37°C to an OD₆₀₀ of 1.5, and the cells were then collected and assayed for β-galactosidase activity. (D) Quantitative comparison of biofilm formation by wild-type strain PAO1 and corresponding mutants.