C-di-GMP hydrolysis by Pseudomonas aeruginosa HD-GYP phosphodiesterases: analysis of the reaction mechanism and novel roles for pGpG

Abstract

In biofilms, the bacterial community optimizes the strategies to sense the environment and to communicate from cell to cell. A key player in the development of a bacterial biofilm is the second messenger c-di-GMP, whose intracellular levels are modulated by the opposite activity of diguanylate cyclases and phosphodiesterases. Given the huge impact of bacterial biofilms on human health, understanding the molecular details of c-di-GMP metabolism represents a critical step in the development of novel therapeutic approaches against biofilms. In this study, we present a detailed biochemical characterization of two c-di-GMP phosphodiesterases of the HD-GYP subtype from the human pathogen Pseudomonas aeruginosa, namely PA4781 and PA4108. Upstream of the catalytic HD-GYP domain, PA4781 contains a REC domain typical of two-component systems, while PA4108 contains an uncharacterized domain of unknown function. Our findings shed light on the activity and catalytic mechanism of these phosphodiesterases. We show that both enzymes hydrolyse c-di-GMP in a two-step reaction via the linear intermediate pGpG and that they produce GMP in vitro at a surprisingly low rate. In addition, our data indicate that the non-phosphorylated REC domain of PA4781 prevents accessibility of c-di-GMP to the active site. Both PA4108 and phosphorylated PA4781 are also capable to use pGpG as an alternative substrate and to hydrolyse it into GMP; the affinity of PA4781 for pGpG is one order of magnitude higher than that for c-di-GMP. These results suggest that these enzymes may not work (primarily) as genuine phosphodiesterases. Moreover, the unexpected affinity of PA4781 for pGpG may indicate that pGpG could also act as a signal molecule in its own right, thus further widening the c-di-GMP-related signalling scenario.

Figure 1. Effect of the overexpression of PA4108 on c-di-GMP levels in E. coli.

(A) Comparison of c-di-GMP levels of the E. coli AB1548 mutant strain, lacking the endogenous PDE YhjH, containing only the pET28 plasmid (control strain) and those of the AB1548 strain complemented with PA4108 (AB1548(∆yhjH) PA4108 in the figure). c-di-GMP levels of the parental E. coli strain (AB472) were used as reference (100%). Data are the mean of at least two independent experiments, each being done in duplicate. (B) PDE activity in soluble cell extracts of BL21(DE3) E. coli strain overexpressing PA4108 (BL21 PA4108 in the figure), analysed after 3.5 h of incubation with 10 µM c-di-GMP. The control strain (BL21 pET28 in the figure) was analyzed after incubation with or without c-di-GMP, in order to evaluate the levels of unreacted c-di-GMP to be compared to the physiological levels of this background. Data are the mean of at least three independent experiments, each being done in duplicate. In (A) and (B) statistical significance with respect to E. coli AB472 or BL21 pET28 with c-di-GMP, respectively, is indicated with an asterisk (p < 0.01).

Figure 2. c-di-GMP hydrolytic activity of PA4108.

(A) Representative time-courses of pGpG and GMP formation obtained after incubation of 0.64 µM PA4108 with 150 µM c-di-GMP (in the presence of 10 mM MgCl₂ and 2.5 mM MnCl₂). The nucleotide content of the reaction mixture was analyzed by RP-HPLC at different times (30, 60, 100, 180 min, at 30°C; solid lines in the figure) and compared with a calibration curve of standard solutions of GMP and pGpG (the chromatogram of a 5 µM solution of both nucleotides is reported as reference, dashed lines). (B) Plot of the initial rate of c-di-GMP hydrolysis (µM of c-di-GMP consumed/min) measured at different c-di-GMP concentrations (black circles). Data were fitted with the Michaelis-Menten equation (continuous line) in order to extrapolate the K_M and V_max parameters (20 µM and 5.8x10^-3 µM/min, respectively). Data are the average of at least two independent experiments.

Figure 3. Effect of the overexpression of PA4781 on c-di-GMP levels in E. coli.

(A) Comparison of c-di-GMP levels of the E. coli AB1548 mutant strain, lacking the endogenous PDE YhjH, containing only the pET28 plasmid (control strain) and those of the AB1548 strain complemented with either PA4781 or PA4781_HD-GYP (a truncated version of the protein lacking the REC domain). c-di-GMP levels of the parental E. coli strain (AB472) were used as reference (100%). Data are the mean of at least two independent experiments, each being done in duplicate. (B) PDE activity in soluble cell extracts of BL21(DE3) E. coli strain overexpressing either PA4781 or PA4781_HD-GYP. The control strain (BL21 pET28 in the figure) was analysed after incubation with or without c-di-GMP, in order to evaluate the levels of unreacted c-di-GMP to be compared to the physiological levels in this background. Data are the mean of at least three independent experiments, each being done in duplicate. In (A) and (B) statistical significance with respect to E. coli AB472 or BL21 pET28 with c-di-GMP, respectively, is indicated with one asterisk (p < 0.01).

Figure 4. c-di-GMP hydrolytic activity of PA4781.

(A) Representative time-courses of pGpG and GMP formation obtained after incubation of 5 µM phosphorylated PA4781 with 120 µM c-di-GMP (in the presence of 10 mM MgCl₂). The nucleotide content of the reaction mixture was analyzed by RP-HPLC at different times (5, 10, 15 min, at 30°C; solid lines in the figure) and compared with a calibration curve of standard solutions of GMP and pGpG (the chromatogram of a 5 µM solution of both nucleotides is reported as reference, dashed lines). (B) Plot of the initial rate of c-di-GMP hydrolysis (µM of c-di-GMP consumed/min) measured at different c-di-GMP concentrations (black circles). Data were fitted with the Michaelis-Menten equation (K_M ~120 µM; V_max=6x10^-2 µM/min). Data are the average of at least two independent experiments.

Figure 5. Predicted interaction of c-di-GMP with PA4108.

(A) Model of the active site of PA4108 in complex with c-di-GMP. Residues in parenthesis indicate amino acid substitutions observed in PA4781. (B) The interaction of PA4108 with the substrate is represented.

Figure 6. c-di-GMP hydrolytic activity of the PA4781 E314A mutant.

(A) Representative time-courses of pGpG and GMP formation obtained after incubation of 5 µM phosphorylated PA4781 E314A with 10 µM c-di-GMP (in the presence of 10 mM MgCl₂); the nucleotide content of the reaction mixture was analyzed by RP-HPLC at different times (5, 10, 15 min, at 30°C; solid lines in the figure) and compared with a calibration curve of standard solutions of GMP and pGpG (the chromatogram of a 5 µM solution of both nucleotides is reported as reference, dashed lines). (B) Plot of the initial rate of c-di-GMP hydrolysis (µM of c-di-GMP consumed/min) measured at different c-di-GMP concentrations (black circles). Data were fitted with the Michaelis-Menten equation (K_M ~7 µM; V_max=1.9x10^-2 µM/min). Data are the average of at least two independent experiments.

Figure 7. pGpG hydrolytic activity of (A) PA4781, (B) PA4781 E314A, (C) PA4108.

For each protein, the initial rate of pGpG hydrolysis (µM of pGpG consumed/min) measured at different pGpG concentrations (black circles) was plotted as a function of µM pGpG. Data were fitted with the Michaelis-Menten equation (continuous line). It should be mentioned that the K_M of PA4108 for pGpG could be an “apparent” value due to the presence of ^~30% of c-di-GMP still bound to the protein. Data are the average of at least two independent experiments.