期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2015
卷号：112
期号：36
页码：E5048-E5057
DOI：10.1073/pnas.1507245112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceCyclic-di-GMP (c-di-GMP) is a ubiquitous bacterial second messenger that regulates complex behaviors such as biofilm formation. These behaviors are changed by altering the intracellular concentration of c-di-GMP. Degradation of c-di-GMP occurs by a two-step process in which one set of phosphodiesterases (PDE-As) linearize the molecule into 5'-phosphoguanylyl-(3',5')-guanosine (pGpG), followed by hydrolysis by unidentified phosphodiesterases (PDE-Bs) into two GMPs. High levels of pGpG inhibit PDE-As, and thus PDE-B activity is important in maintaining c-di-GMP homeostasis. However, the identity of the PDE-B(s) remained unknown. Using a high-throughput binding screen, we identify oligoribonuclease (Orn) as a putative PDE-B. We demonstrate that Orn is the primary source of PDE-B activity in Pseudomonas aeruginosa. Identification of Orn as the primary PDE-B completes the c-di-GMP signaling pathway. 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 {triangleup}orn strains of P. aeruginosa PA14 for pGpG stability. The lysates from {triangleup}orn showed 25-fold decrease in pGpG hydrolysis. Complementation with WT, but not active site mutants, restored hydrolysis. Accumulation of pGpG in the {triangleup}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 {triangleup}orn strain in a pel-dependent manner. Finally, we directly detect elevated pGpG and c-di-GMP in the {triangleup}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.
