title: Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa.
abstract: The second messenger cyclic diguanylate (c-di-GMP) controls diverse cellular processes among bacteria. Diguanylate cyclases synthesize c-di-GMP, whereas it is degraded by c-di-GMP-specific phosphodiesterases (PDEs). Nearly 80% of these PDEs are predicted to depend on the catalytic function of glutamate-alanine-leucine (EAL) domains, which hydrolyze a single phosphodiester group in c-di-GMP to produce 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). However, to degrade pGpG and prevent its accumulation, bacterial cells require an additional nuclease, the identity of which remains unknown. Here we identify oligoribonuclease (Orn)-a 3'→5' exonuclease highly conserved among Actinobacteria, Beta-, Delta- and Gammaproteobacteria-as the primary enzyme responsible for pGpG degradation in Pseudomonas aeruginosa cells. We found that a P. aeruginosa Δorn mutant had high intracellular c-di-GMP levels, causing this strain to overexpress extracellular polymers and overproduce biofilm. Although recombinant Orn degraded small RNAs in vitro, this enzyme had a proclivity for degrading RNA oligomers comprised of two to five nucleotides (nanoRNAs), including pGpG. Corresponding with this activity, Δorn cells possessed highly elevated pGpG levels. We found that pGpG reduced the rate of c-di-GMP degradation in cell lysates and inhibited the activity of EAL-dependent PDEs (PA2133, PvrR, and purified recombinant RocR) from P. aeruginosa. This pGpG-dependent inhibition was alleviated by the addition of Orn. These data suggest that elevated levels of pGpG exert product inhibition on EAL-dependent PDEs, thereby increasing intracellular c-di-GMP in Δorn cells. Thus, we propose that Orn provides homeostatic control of intracellular pGpG under native physiological conditions and that this activity is fundamental to c-di-GMP signal transduction.
authors: Center for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel;
authors: Dorit
authors: D
authors: Cohen
authors: Unité de Biochimie des Interactions Macromoléculaires, Département de Biologie Structurale et Chimie, Institut Pasteur, 75724 Paris Cedex 15, France;
authors: Undine
authors: U
authors: Mechold
authors: Center for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel;
authors: Hadas
authors: H
authors: Nevenzal
authors: Center for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel;
authors: Yafit
authors: Y
authors: Yarmiyhu
authors: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4;
authors: Trevor E
authors: TE
authors: Randall
authors: Department of Medical Microbiology, University of Manitoba, Winnepeg, MB, Canada R3E 0J9;
authors: Denice C
authors: DC
authors: Bay
authors: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4;
authors: Jacquelyn D
authors: JD
authors: Rich
authors: Department of Microbiology, University of Washington, Seattle, WA 98105;
authors: Matthew R
authors: MR
authors: Parsek
authors: Research Core Unit Metabolomics, Institute of Pharmacology, Hannover Medical School, Hannover 30625, Germany.
authors: Volkhard
authors: V
authors: Kaever
authors: Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4; jjharris@ucalgary.ca ehud.banin@biu.ac.il.
authors: Joe J
authors: http://orcid.org/0000-0003-0368-9539
authors: JJ
authors: Harrison
authors: Center for Advanced Materials and Nanotechnology, The Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat-Gan 5290002, Israel; jjharris@ucalgary.ca ehud.banin@biu.ac.il.
authors: Ehud
authors: E
authors: Banin
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D015153:Blotting, Western; 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*; D015964:Gene Expression Regulation, Bacterial; D006706:Homeostasis; D009154:Mutation; D010727:Phosphoric Diester Hydrolases / Q000235:genetics / Q000378:metabolism; D019761:Phosphorus-Oxygen Lyases / Q000235:genetics / Q000378:metabolism; D011550:Pseudomonas aeruginosa / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction
chemical_list: D001426:Bacterial Proteins; D003848:Deoxyguanine Nucleotides; D029968:Escherichia coli Proteins; C034299:deoxydiguanosine diphosphosphate; C062025:bis(3',5')-cyclic diguanylic acid; D005095:Exoribonucleases; C094587:oligoribonuclease; D010727:Phosphoric Diester Hydrolases; D019761:Phosphorus-Oxygen Lyases; C060720:diguanylate cyclase; D006152:Cyclic GMP
keywords: EAL domain; Pseudomonas aeruginosa; biofilm; cyclic diguanylate; oligoribonuclease