Posts Tagged ‘PDE-B’

Biofilm – a memory?

Larry H. Bernstein, MD, FCAP, Curator



New discovery: An enzyme shut off biofilm formation completely

Biofilm, Pseudomonas aeruginosa, Biofilm growth

Biofilms are responsible for the transmission and persistence of human diseases associated with inert surfaces, including medical devices for internal or external use. Biofilm infections are difficult to eradicate, leading to severe clinical complications. A recent publication made a breakthrough finding that an enzyme called oligoribonuclease (Orn) can completely shut off the signaling pathway of biofilm growth. It’s well known that cyclic-di-GMP (c-di-GMP) is the signaling molecule that activates the biofilm formation. The degradation of c-di-GMP involves two steps. The first step is to linearize c-di-GMP to pGpG by PDE-As and the second step is to hydrolyze pGpG into two GMPs by PDE-Bs. The publication reported that Orn is the primary enzyme of PDE-Bs turning pGpG into GMP in the cell, thus completely switch off the biofilm formation. This study is done in Pseudomonas aeruginosa, but may apply to many other bacterial strains that have genetic and physiological similarities with P. aeruginosa. This finding could facilitate the development of new treatment for biofilms and make biofilm-related complications a distant memory.

Mona W. Orr, etc. (September 2015) Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover PNAS


Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover

Mona W. Orra,b,cGregory P. Donaldsona,cGeoffrey B. SeverindJingxin WangeHerman O. SintimeChristopher M. Watersf, and Vincent T. Leea,c,1
NAS Sept 8, 2015; 112(36):  E5048–E5057               

Cyclic-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 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-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 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-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.


Cyclic-di-GMP (c-di-GMP) is a phylogenetically widely used bacterial second messenger (1). Cyclic-di-GMP is synthesized by diguanylate cyclase enzymes (DGC) that contain the GGDEF domain (2, 3). Once synthesized, the c-di-GMP binds to intracellular receptors to decrease motility and increase biofilm formation, contributing to the virulence of several pathogens (1, 47). Cyclic-di-GMP signaling is terminated by phosphodiesterases (PDE-A) that linearize it into pGpG, which is then hydrolyzed into GMP by an unknown phosphodiesterase termed PDE-B (2). Bacteria encode two structurally unrelated PDE-As: one containing the EAL domain (810) and a second containing the HD-GYP domain (11). The overexpression of GGDEF domain DGCs elevates c-di-GMP and c-di-GMP–regulated processes (8, 12); conversely, overexpression of the EAL and HD-GYP domain PDE-As decreases c-di-GMP and c-di-GMP–regulated processes (8, 1315). These c-di-GMP synthesizing and degrading domains are commonly linked to sensory and signal transduction domains (1, 16), thereby allowing synthesis and degradation of c-di-GMP in response to environmental changes.

In addition to regulation by extracellular signals, c-di-GMP homeostasis is subject to feedback inhibition. Crystal structures of the DGCs PleD from Caulobacter crescentus and WspR from Pseudomonas aeruginosa show that c-di-GMP binds to an RxxD domain I-site to inhibit c-di-GMP synthesis (17, 18). Additionally, the Xanthomonas campestris XCC4471 DGC lacking an RxxD motif can also be inhibited by excess c-di-GMP through c-di-GMP binding to and occluding the active site (19). The linearized c-di-GMP hydrolysis product, 5ʹ-phosphoguanylyl-(3ʹ,5ʹ)-guanosine (pGpG), also plays an active role in cyclic dinucleotide turnover. Purified YfgF from E. coli, a PDE-A, is inhibited by excess pGpG through an unknown mechanism (20). Here, we show that the EAL domain PDE-A RocR from the P. aeruginosa PA14 strain is also inhibited by excess pGpG via direct competition with c-di-GMP binding in the active site. Accordingly, the addition of excess pGpG results in an increased c-di-GMP half-life in vitro, suggesting that removal of pGpG is required for terminating c-di-GMP signaling.

To elucidate the mechanism of c-di-GMP signal termination, we sought to identify the PDE-B(s) responsible for cleaving pGpG. Previously, HD-GYP domain-containing proteins were proposed to be the PDE-Bs involved in pGpG hydrolysis because they bind pGpG with higher affinity than c-di-GMP (21). However, HD-GYP domain proteins are missing in some genomes that contain other c-di-GMP signaling machinery (1), suggesting that other enzymes must be responsible for PDE-B activity. To identify PDE-B(s), we used a screen based on the differential radial capillary action of ligand assay (DRaCALA) (22, 23) to probe an ORF library of Vibrio cholerae El Tor N16961 (24) for proteins that bind pGpG. This screen identified oligoribonuclease (Orn) as a protein that binds pGpG, but not c-di-GMP. Orn is an exoribonuclease that cleaves two- to five-nucleotide-long RNA molecules (25). We found that purified Orn from both V. choleraeand P. aeruginosa bound and cleaved pGpG. To determine whether Orn is the primary PDE-B in bacteria, we show that whole cell lysates of an orn transposon mutant (orn::tn) from the P. aeruginosa PA14 Non-Redundant Transposon Insertion Mutant Library (26) and an in-frame deletion mutant of orn (∆orn) were decreased in pGpG cleaving activity by 25-fold compared with the parental strain. Complementation with WT orn, but not active site point mutants, restored pGpG hydrolysis in cell lysates. Thus, loss of orn is expected to increase pGpG concentration in vivo and may inhibit PDE-A activity to also elevate c-di-GMP concentrations in the ∆orn strain. In support of elevated c-di-GMP in the ∆orn strain, we observed three times more activity from the c-di-GMP–responsive pel promoter FleQ (27). We also demonstrate that two c-di-GMP–governed phenotypes, biofilm formation (28) and aggregation (29), are enhanced in the ∆orn strain and are dependent on the c-di-GMP–regulated PEL exopolysaccharides (30). Using LC-MS/MS, we directly detect higher levels of both pGpG and c-di-GMP in extracts of the ∆orn strain compared with WT. Taken together, these results indicate that Orn is the primary PDE-B responsible for degrading pGpG in P. aeruginosa.

Oligoribonucleases as PDE-Bs.

Early characterization of c-di-GMP from the Benziman laboratory identified that c-di-GMP is linearized by PDE-As into pGpG, which is then degraded into two GMPs through the action of a presumed PDE-B (44). Through nearly three decades of c-di-GMP research that followed, the identity of the PDE-B remained unknown. We demonstrate here that the 3′-5′exoribonuclease Orn is the primary PDE-B in P. aeruginosa. The linearized product of c-di-GMP hydrolysis by PDE-As, pGpG, is a two-nucleotide-long RNA that matches the known substrates of Orn. We present several lines of evidence indicating that Orn is the primary PDE-B for c-di-GMP in P. aeruginosa: (i) Orn binds pGpG and not c-di-GMP, (ii) Orn rapidly degrades pGpG in vitro, (iii) lysates of P. aeruginosa orn mutants are greatly reduced for PDE-B activity, (iv) DEDDh catalytic residues used to degrade oligonucleotides are required for pGpG cleavage, and (v) loss of orn results in enhanced intracellular pGpG and c-di-GMP. This additional role for Orn is important because we demonstrate that the loss of Orn results in increased c-di-GMP and c-di-GMP governed phenotypes, likely due to accumulated pGpG inhibiting PDE-As as shown in our model (Fig. 7A).

Fig. 7.

The distribution of RNases, GGDEF domain proteins, and diadenylate cyclase domain proteins. (A) A model showing the synthesis and degradation of c-di-GMP with the inclusion of pGpG-mediated feedback inhibition of EAL-domain PDE-As and Orn as a PDE-B. (B) A simplified phylogenetic tree of prokyaryotic and archaeal species from the COG database and a table showing the number of species from each class that encodes the following proteins: Orn, NrnA, NrnB, GGDEF domains, and diadenylate cyclase (DisA) domains. The number of species were determined by a COG database search: Orn = COG1949; NrnA = COG0618; NrnB = COG2404; GGDEF = COG2199, COG3706; DisA = COG1623, COG1624.

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