Characterization and analysis of a novel diguanylate cyclase PA0847 from Pseudomonas aeruginosa PAO1

Abstract

Background: As a central signaling molecule, cyclic diguanylate (c-di-GMP) is found to regulate various bacterial phenotypes, especially those involved in pathogen infection and drug resistance. Noticeably, many microbes have up to dozens of proteins that are involved in c-di-GMP metabolism. This apparent redundancy and the relevant functional specificity have become the focus of research. While a number of these proteins have been identified and investigated, the functions of PA0847, a PAS and GGDEF domain-containing protein from Pseudomonas aeruginosa PAO1, remain unclear. Materials and methods: In the current study, microbiology, biochemistry and structural biology methods were applied to characterize the gene/protein of PA0847. Results: We showed that PA0847 affects bacterial motility but not biofilm formation. We recorded the phenotypic influences of amino acids and compounds, and found that PA0847 is involved in response to various environmental nutrients and factors, suggesting its possible role in sensing environmental cues. Both in-vitro and in-vivo studies showed that PA0847 is an active diguanylate cyclase (DGC), whose activity depends on the neighboring PAS domain. Interestingly, PA0847 demonstrates no significant product inhibition, though the key residues of two I-sites for c-di-GMP binding are conserved in its GGDEF domain. A local structural change imposed by an adjacent tyrosine residue was identified, which indicates the structural and functional diversities of the GGDEF family proteins. Conclusion: Our data provide evidence for understanding the signaling mechanism of the unique c-di-GMP metabolizing protein PA0847.

Keywords: GGDEF domain; Pseudomonas aeruginosa; c-di-GMP; diguanylate cyclase; structure.

Figure 1

The gene of PA0847 affects biofilm formation and motility phenotypes in Pseudomonas aeruginosa. Notes: (A) Swimming motility of P. aeruginosa PAO1 (WT), the PA0847 mutants and the strains complemented with empty vector or PA0847, colony morphologies taken after 20–24 hours of incubation at 37°C, and swimming motility assays performed on medium supplemented with 0.3% agar. (B) Swarming motility of P. aeruginosa PAO1 (WT), the PA0847 mutants and the strains complemented with empty vector or PA0847, colony morphologies taken after 36–48 hours of incubation at 37°C, and swarming motility assays performed on medium supplemented with 0.45% agar. (C) Statistical analysis of biofilms of P. aeruginosa PAO1, the PA0847 mutant or the strains complemented with empty vector or PA0847. The biofilm data shown are mean ± SD of at least three replicates. PAO1: P. aeruginosa PAO1 WT; ΔPA0847-1 or ΔPA0847-2: two PA0847 transposon mutant strains; PAO1/pACYC184: PAO1 harboring the empty vector pACYC184; ΔPA0847/pACYC184: ΔPA0847-1 or ΔPA0847-2 harboring empty vector pACYC184; ΔPA0847-1/PA0847 or ΔPA0847-2/PA0847: ΔPA0847-1 or ΔPA0847-2 mutant strains harboring full-length PA0847 with its native promoter and RBS cloned into pACYC184. Abbreviation: WT, wild type.

Figure 2

Pseudomonas aeruginosa PAO1 and PA0847 mutants motility and biofilm formation in response to individual amino acids differently. Notes: (A) Differential impacts of individual amino acids on bacterial motility. Swimming motility of both P. aeruginosa PAO1 and PA0847 mutants in response to five amino acids. Shown are pictures of plates incubated for 20–24 hours at 30°C. Left: wild type, upper right: ΔPA0847-1, lower right: ΔPA0847-2. (B) P. aeruginosa biofilm formation in response to individual amino acids. Shown is the quantification of 24 hour biofilms formed in the tubes and stained with Crystal Violet, solubilized in 95% ethanol and measured at 595 nm. Asterisks indicate values significantly different from the wild type. ***p<0.005.

Figure 3

Diguanylate cyclase activity of PA0847 GGDEF domain. Notes: (A) Molecular architecture of PA0847. Domains are predicted by using the Conserved-Domain Database search from NCBI (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and transmembrane helices are predicted in the TMHMM Server (http://www.cbs.dtu.dk/services/TMHMM/). The amino acid number of each domain is indicated above the corresponding bar. (B) DGC activity of PA0847 GGDEF domain is detected by HPLC assays. GTP and c-di-GMP standard HPLC profiles are indicated above the assays. (C) The binding of GTPγS to PAS-GGDEF domain showed a binding affinity (K_d) of 15.3±1.3 µM. (D) Gel filtration profiles of PAS-GGDEF and GGDEF. The calculated MW of each peak based on protein standards is indicated above the peak. (E) Congo Red staining of wild-type PAO1 and ΔPA0847.

Figure 4

Structural model of PA0847 GGDEF dimer. Notes: (A) Sequence alignment between PA0847 GGDEF domain and other DGCs of known structure. Secondary structure elements are indicated above the sequences. Key residues involved in binding GTP are highlighted in yellow and putative residues in inhibition sites are highlighted in grey. (B) Structural model of PA0847 GGDEF dimer in complex with GTPs and Mg²⁺. (C) GTP binding residues labeled and depicted as stick models. (D) c-di-GMP synthesis by wild type and GGDEF point mutants of PA0847. (E) Binding of c-di-GMP to PAS-GGDEF dual domain. (F) Structural comparison of GGDEF domains between PA0847 (green) and PleD (pink). Bulky residue Y⁷⁰⁰ was showed in stick. (G) Different phenotype of Escherichia coli with overexpressed wild type and mutants of PAS-GGDEF domains.