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：11359-11364
DOI：10.1073/pnas.1421450112
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：SignificanceMany bacteria possess enzymes that synthesize and degrade the intracellular second messenger cyclic diguanylate (c-di-GMP). Bacteria use this molecule to relay environmental signals into physiological responses that control motility, virulence, and biofilm formation. There are two pathways for enzymatic c-di-GMP degradation. One of these pathways involves the production of an intermediate molecule called 5'-phosphoguanylyl-(3',5')-guanosine (pGpG). Although many enzymes responsible for c-di-GMP degradation have been characterized, microbiologists have long sought those responsible for pGpG degradation. Here we identify that oligoribonuclease (Orn) mediates pGpG degradation and show that Orn is important for c-di-GMP signaling in the human pathogen Pseudomonas aeruginosa. This discovery reveals that nanoribonucleases, which have been considered housekeeping proteins crucial for mRNA turnover, also have a key role in c-di-GMP signaling. 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 {Delta}orn 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, {Delta}orn 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 {Delta}orn 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.
