Regulation of Exopolysaccharide Production by ProE, a Cyclic-Di-GMP Phosphodiesterase in Pseudomonas aeruginosa PAO1

Abstract

The ubiquitous second messenger c-di-GMP is involved in regulation of multiple biological functions including the important extracellular matrix exopolysaccharides (EPS). But how c-di-GMP metabolic proteins influence EPS and their enzymatic properties are not fully understood. Here we showed that deletion of proE, which encodes a protein with GGDEF-EAL hybrid domains, significantly increased the transcriptional expression of the genes encoding EPS production in Pseudomonas aeruginosa PAO1 and changed the bacterial colony morphology. Our data showed that ProE is a very active phosphodiesterase (PDE), with a high enzyme activity in degradation of c-di-GMP. Interestingly, the optimal activity of ProE was found in the presence of Co²⁺, unlike other PDEs that commonly rely on Mg²⁺ or Mn²⁺ for best performance. Furthermore, we identified three widely conserved novel residues that are critical for the function of ProE through site-directed mutagenesis. Subsequent study showed that ProE, together with other three key PDEs, i.e., RbdA, BifA, and DipA regulate the EPS production in P. aeruginosa PAO1. Moreover, by using the GFP-fusion approach, we observed that these four EPS associated-PDEs showed a polar localization pattern in general. Taken together, our data unveil the molecular mechanisms of ProE in regulation of EPS production, and provide a new insight on its enzymatic properties in degradation of c-di-GMP.

Keywords: Pel and Psl; Pseudomonas aeruginosa; c-di-GMP; enzymatic properties; exopolysaccharide; phosphodiesterase.

FIGURE 1

The proE mutant showed a wrinkly colony morphology. (A) Genetic organization and domain structures of ProE, and the active site of the GGDEF domain is degenerate. (B) Mutation of proE affected the colony morphology on Congo-Red plate after growth on 25°C for 2 days, nor does mutation of PA5294 have the similar phenotype. Scale bar = 2 mm. (C) Growth curves of PAO1 and ΔproE. The data are means of three replicates and error bars indicate standard deviation.

FIGURE 2

ProE regulated EPS production was dependent on pel and psl. (A) The wrinkly colony is only abolished when both pelA and pslA are mutated in the ΔfleQ background, while a pslA mutation is not sufficient to confer smooth colony morphology in the ΔproE background. All strains were growth on Congo-Red plates. (B) The relative gene expression of pelA and pslA in PAO1, ΔproE, ΔfleQ, Δ proE (proE),ΔfleQ (fleQ),ΔpelA, and ΔpslA by qRT-PCR analysis. The data are means of three replicates and error bars indicate standard deviation. *P < 0.05; **P < 0.01; ***P < 0.0001. (C) Complementation of proE can restore the wrinkly colony phenotype of ΔfleQ, and complementation of fleQ can also substantially alleviate the wrinkly colony phenotype of ΔproE. Scale bar = 2 mm.

FIGURE 3

Sequence alignment of EAL-containing phosphodiesterases. The amino acid sequence of RocR (Q9HX69), ProE (Q9HTQ9), RbdA (Q9I580), BifA (Q9HW35) from P. aeruginosa, TBD1265 (Q3SJE6), TBD1269 (Q3SJE2) from Thiobacillus denitrificans, CV0542 (Q7P0M4), CV2505 (Q7NV41) from Chromobacterium violaceum, SO2039 (Q8EFE2) from Shewanella oneidensis, YahA (P21514) from Escherichia coli, NE0566 (Q82WU5) from Nitrosomonas europaea. The amino acids highlighted with black stands for 100% similarity, and gray indicates similarity level ≥75%. E³²⁸, N³⁸⁷, E⁴¹⁹, D⁴⁴⁹, D⁴⁵⁰, E⁵⁰⁶ (filled triangle) are conserved residues which may bind to metal ion (Tamayo et al., 2005; Rao et al., 2008; Tchigvintsev et al., 2010; Yang et al., 2017). The conserved loop 6 (DFG(T/A)GYSS) is essential for the dimerization of EAL domain and Mg²⁺, c-di-GMP binding (Rao et al., 2009). R³³², N³⁸⁷ are (asterisks) reported to bind with substrate (Rao et al., 2008; Tchigvintsev et al., 2010; Yang et al., 2017). K⁴⁷⁰ (filled circle) is essential for coordinating with water molecule for catalysis and interacting with residue E⁴¹⁹ residue (Rao et al., 2008; Tchigvintsev et al., 2010; Yang et al., 2017). Q⁵²⁶ is reported to interact with E³²⁸ and K⁴⁷⁰ (open circle) (Tchigvintsev et al., 2010; Yang et al., 2017).

FIGURE 4

Purified ProE is an active phosphodiesterase. (A,C,E) The standard of c-di-GMP (A), pGpG (C), GTP (E) in a final concentration of 100 μM was prepared in the reaction buffer, and 10 μl was injected for HPLC analysis. (B) RocR and (D) ProE after incubation with c-di-GMP at a final concentration of 50 μM at 37°C for 20 min. (F) ProE incubated with 50 μM GTP for 5 min at room temperature prior to addition of c-di-GMP, then the enzyme activity was analysis by HPLC. (G) Quantification of the enzyme activity of ProE and RocR. (H) Quantification of the enzyme activity of ProE and ProE with GTP prior to addition substrate. (I) The influence of pH on enzyme activity. The pH of the phosphodiesterase reaction was adjusted to 3–12 prior to the addition of enzyme and substrate (37°C, 5 mM MgCl₂). ProE activity was higher in alkaline condition. (J) The influence of temperature on enzyme activity. The ProE phosphodiesterase reaction was performed at a range of temperatures from 10°C to 90°C (pH 8.0, 5 mM MgCl₂). The highest rate of c-di-GMP hydrolysis was achieved at 60°C and 70°C. At temperatures lower than 30°C, no activity was detectable. (K) Dependence of the phosphodiesterase activity of ProE on divalent metal cations. Reaction buffer (37°C, pH 8.0) The data are means of three replicates and error bars indicate standard deviation. *P < 0.05; **P < 0.01, ***P < 0.0001.

FIGURE 5

Functional analysis of the conserved residues of EAL domain of ProE. (A) After site-directed mutagenesis, the enzyme activity of mutant proteins were analyzed. The data are means of three replicates and error bars indicate standard deviation. (B) Effect of conserved residues on the colony morphology of P. aeruginosa strain PAO1 and its derivatives on Congo-Red plates. The native proE gene is deleted, and the proE alleles are according to complementation of mutated pBBR1-MCS5-proE into ΔproE. Scale bar = 2 mm.

FIGURE 6

Homology models of the ProE with PA3825 and RmcA. Superimposition of the wild type ProE models with templates of 4y9m (PA3825-Apo), RMSD = 0.064 (214 to 214 atoms) (A), and 4y9p (PA3825-Ca²⁺ and c-di-GMP), RMSD = 0.04 (213 to 213 atoms) (B), and 5m3C (RmcA-Ca²⁺-GTP), RMSD = 0.190 (721 to 721 atoms) (C). ProE and templates are shown in lightblue and orange cartoons, respectively. C-di-GMP and GTP are shown as yellow sticks, calcium ions are shown in yellow spheres. (D) Superposition of Apo-ProE and ligand-bound ProE models reveals conformational changes due to ligand interaction. Apo-ProE and ligand-bound ProE are shown in lightblue and lime cartoons, respectively. The number represents the distance between residues (Å).

FIGURE 7

Mapping of conserved residues identified in ProE with homolog model. (A) The surface structure showing the localization of the conserved residues in the ProE model around the calcium ions and c-di-GMP. (B) The labeled residues indicate those interact with the calcium ions in ProE. (C) The labeled residues indicate those form direct or indirect polar contacts with c-di-GMP. (D) The residues E509 and Y528 interact with S531 and E509, respectively, from another monomer to allow dimer formation. (E) Interaction of conserved residues K470 and E422 with their adjacent residues. (F) Interaction of K470A and E422A with their adjacent residues. Calcium ions and c-di-GMP molecules are shown as yellow spheres and sticks, respectively. Polar interaction are shown as yellow dashes.

FIGURE 8

Functional and subcellular localization analysis of EPS-associated phosphodiesterases. (A) ProE and other three phosphodiesterases synergically regulated the colony morphology in P. aeruginosa. Deletion of either proE, rbdA, bifA or dipA can cause wrinkly morphology at different extent, and the functional defect can be restored by each other. Scale bar = 2 mm. (B) Subcellular localization of GFP fusion proteins. Bacterial colonies expressing indicated GFP fusion proteins were picked from overnight LB agar plates, resuspended in PBS and 2 μl culture was spotted onto a glass slide coated with 1% agarose. Images were taken by using epifluorescent microscopy. Scale bar = 10 μM.