title: Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover.
abstract: The bacterial second messenger cyclic di-GMP (c-di-GMP) controls biofilm formation and other phenotypes relevant to pathogenesis. Cyclic-di-GMP is synthesized by diguanylate cyclases (DGCs). Phosphodiesterases (PDE-As) end signaling by linearizing c-di-GMP to 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), which is then hydrolyzed to two GMP molecules by yet unidentified enzymes termed PDE-Bs. We show that pGpG inhibits a PDE-A from Pseudomonas aeruginosa. In a dual DGC and PDE-A reaction, excess pGpG extends the half-life of c-di-GMP, indicating that removal of pGpG is critical for c-di-GMP homeostasis. Thus, we sought to identify the PDE-B enzyme(s) responsible for pGpG degradation. A differential radial capillary action of ligand assay-based screen for pGpG binding proteins identified oligoribonuclease (Orn), an exoribonuclease that hydrolyzes two- to five-nucleotide-long RNAs. Purified Orn rapidly converts pGpG into GMP. To determine whether Orn is the primary enzyme responsible for degrading pGpG, we assayed cell lysates of WT and ∆orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from ∆orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the ∆orn strain could inhibit PDE-As, increasing c-di-GMP concentration. In support, we observed increased transcription from the c-di-GMP-regulated pel promoter. Additionally, the c-di-GMP-governed auto-aggregation and biofilm phenotypes were elevated in the ∆orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the ∆orn strain. Thus, we identified that Orn serves as the primary PDE-B enzyme that removes pGpG, which is necessary to complete the final step in the c-di-GMP degradation pathway.
authors: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; Biological Sciences Graduate Program, University of Maryland, College Park, MD 20742; Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742;
authors: Mona W
authors: MW
authors: Orr
authors: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742;
authors: Gregory P
authors: GP
authors: Donaldson
authors: Department of Biochemistry and Molecular Biology, Michigan State University, East Lansing, MI 48824;
authors: Geoffrey B
authors: GB
authors: Severin
authors: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742;
authors: Jingxin
authors: J
authors: Wang
authors: Department of Chemistry and Biochemistry, University of Maryland, College Park, MD 20742;
authors: Herman O
authors: HO
authors: Sintim
authors: Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824.
authors: Christopher M
authors: CM
authors: Waters
authors: Department of Cell Biology and Molecular Genetics, University of Maryland, College Park, MD 20742; Maryland Pathogen Research Institute, University of Maryland, College Park, MD 20742; vtlee@umd.edu.
authors: Vincent T
authors: VT
authors: Lee
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D018441:Biofilms / Q000254:growth & development; D002853:Chromatography, Liquid; D006152:Cyclic GMP / Q000031:analogs & derivatives* / Q000378:metabolism; D003848:Deoxyguanine Nucleotides / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism; D005095:Exoribonucleases / Q000235:genetics / Q000378:metabolism*; D006157:Guanosine Monophosphate / Q000378:metabolism; D006706:Homeostasis; D006868:Hydrolysis; D008969:Molecular Sequence Data; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D011550:Pseudomonas aeruginosa / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; C034299:deoxydiguanosine diphosphosphate; C062025:bis(3',5')-cyclic diguanylic acid; D006157:Guanosine Monophosphate; D005095:Exoribonucleases; C094587:oligoribonuclease; D010727:Phosphoric Diester Hydrolases; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP
keywords: PDE-B; cyclic di-GMP; nanoRNase; oligoribonuclease; pGpG