guanosine-monophosphate and bis(3--5-)-cyclic-diguanylic-acid

Cyclic di-guanosine monophosphate (c-di-GMP) is a second messenger found ubiquitously in bacteria. This signaling molecule regulates a variety of physiological activities such as phototaxis and flocculation in cyanobacteria and is critical for their environmental adaptation. Although genes encoding the enzymes for synthesis and/or degradation of c-di-GMP are found in the genomes of both multicellular and unicellular cyanobacteria, little is known about the biological functions of these enzymes in cyanobacterial cells. Here we have established a robust and highly sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS)-based method for c-di-GMP quantification using a cost-effective solvent, methanol. Quantification methods were validated by measuring c-di-GMP in the cyanobacterium Synechococcus elongatus PCC 7942 through spiking and recovery assays after which the method was applied to examine short-term changes in cellular levels of c-di-GMP in response to a transition from light to dark or from dark to light in S. elongatus. Results showed that a transient increase in c-di-GMP upon transitioning from light to dark was occurring which resembled responses involving cyclic adenosine monophosphate and other second messengers in cyanobacteria. These findings demonstrated that our method enabled relatively specific and sensitive quantification of c-di-GMP in cyanobacteria at lower cost.

Topics: Bacterial Proteins; Chromatography, Liquid; Cyanobacteria; Cyclic GMP; Guanosine Monophosphate; Spectrometry, Mass, Electrospray Ionization; Tandem Mass Spectrometry

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.

Topics: Amino Acid Sequence; Bacterial Proteins; Biofilms; Chromatography, Liquid; Cyclic GMP; Deoxyguanine Nucleotides; Escherichia coli Proteins; Exoribonucleases; Guanosine Monophosphate; Homeostasis; Hydrolysis; Molecular Sequence Data; Mutation; Phosphoric Diester Hydrolases; Phosphorus-Oxygen Lyases; Protein Binding; Pseudomonas aeruginosa; Sequence Homology, Amino Acid; Tandem Mass Spectrometry

Commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-(propargyl)guanosine is converted to its 3'-O-levulinyl ester in a yield of 91%. The reaction of commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine with N(2)-isobutyryl-2'-O-propargyl-3'-O-(levulinyl)guanosine provides, after P(III) oxidation, 3'-/5'-deprotection, and purification, the 2'-O-propargylated guanylyl(3'-5')guanosine 2-cyanoethyl phosphate triester in a yield of 88%. Phosphitylation of this dinucleoside phosphate triester with 2-cyanoethyl tetraisopropylphosphordiamidite and 1H-tetrazole, followed by an in situ intramolecular cyclization, gives the propargylated cyclic dinucleoside phosphate triester, which is isolated in a yield of 40% after P(III) oxidation and purification. Complete removal of the nucleobases, phosphates, and 2'-O-tert-butyldimethylsilyl protecting groups leads to the desired propargylated c-di-GMP diester. Cycloaddition of a biotinylated azide with the propargylated c-di-GMP diester under click conditions provides the biotinylated c-di-GMP conjugate in an isolated yield of 62%. Replacement of the 6-oxo function of N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-3'-O-levulinyl-2'-O-(propargyl)guanosine with a 2-cyanoethylthio group is effected by treatment with 2,4,6-triisopropybenzenesulfonyl chloride and triethylamine to give a 6-(2,4,6-triisopropylbenzenesulfonic acid) ester intermediate. Reaction of this key intermediate with 3-mercaptoproprionitrile and triethylamine, followed by 5'-dedimethoxytritylation, affords the 6-(2-cyanoethylthio)guanosine derivative in a yield of 70%. The 5'-hydroxy function of this derivative is reacted with commercial N(2)-isobutyryl-5'-O-(4,4'-dimethoxytrityl)-2'-O-tert-butyldimethylsilyl-3'-O-[(2-cyanoethyl)-N,N-diisopropylaminophosphinyl]guanosine. The reaction product is then converted to the mono-6-thioated c-di- GMP biotinylated conjugate under conditions highly similar to those described above for the preparation of the biotinylated c-di-GMP conjugate, and isolated in similar yields.

Topics: Azides; Biotinylation; Click Chemistry; Cyclic GMP; Dinucleoside Phosphates; Guanosine; Guanosine Monophosphate; Tetrazoles

Many bacterial pathogens produce diffusible signal factor (DSF)-type quorum sensing (QS) signals in modulation of virulence and biofilm formation. Previous work on Xanthomonas campestris showed that the RpfC/RpfG two-component system is involved in sensing and responding to DSF signals, but little is known in other microorganisms. Here we show that in Burkholderia cenocepacia the DSF-family signal cis-2-dodecenoic acid (BDSF) negatively controls the intracellular cyclic dimeric guanosine monophosphate (c-di-GMP) level through a receptor protein RpfR, which contains Per/Arnt/Sim (PAS)-GGDEF-EAL domains. RpfR regulates the same phenotypes as BDSF including swarming motility, biofilm formation, and virulence. In addition, the BDSF(-) mutant phenotypes could be rescued by in trans expression of RpfR, or its EAL domain that functions as a c-di-GMP phosphodiesterase. BDSF is shown to bind to the PAS domain of RpfR with high affinity and stimulates its phosphodiesterase activity through induction of allosteric conformational changes. Our work presents a unique and widely conserved DSF-family signal receptor that directly links the signal perception to c-di-GMP turnover in regulation of bacterial physiology.

Topics: Bacterial Proteins; Burkholderia cenocepacia; Cell Communication; Cyclic GMP; Dimerization; Fatty Acids, Monounsaturated; Guanosine Monophosphate; Models, Genetic; Mutagenesis; Mutation; Phenotype; Protein Binding; Quorum Sensing; Receptors, Cell Surface; Signal Transduction; Virulence

(3'-5')-Cyclic diguanylate (c-di-GMP) is a bacterial second messenger with immunomodulatory activities in mice suggesting potential applications as a vaccine adjuvant and as a therapeutic agent. Clinical studies in larger animals or humans will require larger doses that are difficult and expensive to generate by currently available chemical or enzymatic synthesis and purification methods. Here we report the production of c-di-GMP at the multi-gram scale from the economical precursors guanosine monophosphate (GMP) and adenosine triphosphate by a "one-pot" three enzyme cascade consisting of GMP kinase, nucleoside diphosphate kinase, and a mutated form of diguanylate cyclase engineered to lack product inhibition. The c-di-GMP was purified to apparent homogeneity by a combination of anion exchange chromatography and solvent precipitation and was characterized by reversed phase high performance liquid chormatography and mass spectrometry, nuclear magnetic resonance spectroscopy, and further compositional analyses. The immunomodulatory activity of the c-di-GMP preparation was confirmed by its potentiating effect on the lipopolysaccharide-induced interleukin 1β, tumor necrosis factor α, and interleukin 6 messenger RNA expression in J774A.1 mouse macrophages.

Topics: Adenosine Triphosphate; Animals; Biotechnology; Cell Line; Chromatography, Ion Exchange; Cloning, Molecular; Cyclic GMP; Escherichia coli; Escherichia coli Proteins; Guanosine Monophosphate; Guanylate Kinases; Immunologic Factors; Interleukin-1beta; Interleukin-6; Lipopolysaccharides; Macrophages; Magnetic Resonance Spectroscopy; Mice; Mutation; Nucleoside-Diphosphate Kinase; Phosphorus-Oxygen Lyases; Recombinant Proteins; Transformation, Bacterial; Tumor Necrosis Factor-alpha

Cyclic diguanylate (c-di-GMP) is a global regulator that modulates pathogen virulence and biofilm formation in bacteria. Although a bioinformatic study revealed that PilZ domain proteins are the long-sought c-di-GMP binding proteins, the mechanism by which c-di-GMP regulates them is uncertain. Pseudomonas putida PP4397 is one such protein that contains YcgR-N and PilZ domains and the apo-PP4397 structure was solved earlier by the Joint Center for Structural Genomics. We determined the crystal structure of holo-PP4397 and found that two intercalated c-di-GMPs fit into the junction of its YcgR-N and PilZ domains. Moreover, c-di-GMP binding induces PP4397 to undergo a dimer-to-monomer transition. Interestingly, another PilZ domain protein, VCA0042, binds to a single molecule of c-di-GMP, and both its apo and holo forms are dimeric. Mutational studies and the additional crystal structure of holo-VCA0042 (L135R) showed that the Arg122 residue of PP4397 is crucial for the recognition of two molecules of c-di-GMP. Thus, PilZ domain proteins exhibit different c-di-GMP binding stoichiometry and quaternary structure, and these differences are expected to play a role in generating diverse forms of c-di-GMP-mediated regulation.

Topics: Amino Acid Sequence; Bacterial Proteins; Binding Sites; Crystallography, X-Ray; Cyclic GMP; Dimerization; Guanosine Monophosphate; Hydrogen Bonding; Kinetics; Models, Molecular; Molecular Sequence Data; Molecular Weight; Protein Binding; Protein Structure, Secondary; Protein Structure, Tertiary; Pseudomonas putida; Sequence Homology, Amino Acid

The bacterial second messenger c-di-GMP is used in many species to control essential processes that allow the organism to adapt to its environment. The c-di-GMP riboswitch (GEMM) is an important downstream target in this signaling pathway and alters gene expression in response to changing concentrations of c-di-GMP. The riboswitch selectively recognizes its second messenger ligand primarily through contacts with two critical nucleotides. However, these two nucleotides are not the most highly conserved residues within the riboswitch sequence. Instead, nucleotides that stack with c-di-GMP and that form tertiary RNA contacts are the most invariant. Biochemical and structural evidence reveals that the most common natural variants are able to make alternative pairing interactions with both guanine bases of the ligand. Additionally, a high-resolution (2.3 A) crystal structure of the native complex reveals that a single metal coordinates the c-di-GMP backbone. Evidence is also provided that after transcription of the first nucleotide on the 3'-side of the P1 helix, which is predicted to be the molecular switch, the aptamer is functional for ligand binding. Although large energetic effects occur when several residues in the RNA are altered, mutations at the most conserved positions, rather than at positions that base pair with c-di-GMP, have the most detrimental effects on binding. Many mutants retain sufficient c-di-GMP affinity for the RNA to remain biologically relevant, which suggests that this motif is quite resilient to mutation.

Topics: Base Pairing; Base Sequence; Cyclic GMP; Guanine; Guanosine Monophosphate; Ligands; Mutation; Protein Structure, Tertiary; Second Messenger Systems

Cyclic ribodiguanylic acid, c-(GpGp), is the endogenous effector regulator of cellulose synthase. Its three-dimensional structure from two different crystal forms (tetragonal and trigonal) has been determined by X-ray diffraction analysis at 1 A resolution. In both crystal forms, two independent c-(GpGp) molecules associate with each other to form a self-intercalated dimer. A hydrated cobalt ion is found to coordinate to two N7 atoms of adjacent guanines, forcing these two guanines to destack with a large dihedral angle (32 degrees), in the dimer of the tetragonal form. This metal coordination mechanism may be relevant to that of the anticancer drug cisplatin. Moreover, c-(GpGp) exhibits unusual spectral properties not seen in any other cyclic dinucleotide. It interacts with planar organic intercalator molecules in ways similar to double helical DNA. We propose a cage-like model consisting of a tetrameric c-(GpGp) aggregate in which a large cavity ('host') is generated to afford a binding site for certain planar intercalators ('guests').

Topics: Cyclic GMP; Guanine Nucleotides; Guanosine Monophosphate; Intercalating Agents; Molecular Structure; Spectrophotometry, Ultraviolet; X-Ray Diffraction