phosphorus-radioisotopes and bis(3--5-)-cyclic-diguanylic-acid

Diguanylate cyclases that synthesize and phosphodiesterases that hydrolyze the second messenger cyclic-di-GMP (c-di-GMP) are at the center of bacterial signaling pathways that control behaviors relevant to all aspects of microbial physiology and pathogenesis (Romling et al., Microbiol Mol Biol Rev 77(1):1-52, 2013). Bioinformatics tools can easily predict the presence of the diguanylate cyclase GGDEF domain, or the EAL and HD-GYP domains associated with phosphodiesterase activity. However, experimental confirmation of enzymatic activity is still necessary, as many proteins contain degenerate domains that lack catalytic activity but nonetheless function as c-di-GMP receptors.

Topics: 3',5'-Cyclic-GMP Phosphodiesterases; Bacteria; Cyclic GMP; Enzyme Activation; Escherichia coli Proteins; Gene Expression; Phosphorus Radioisotopes; Phosphorus-Oxygen Lyases; Recombinant Proteins

Cyclic-di-GMP phosphodiesterases (PDEs) catalyze the hydrolysis of the bacterial second messenger c-di-GMP. This protocol describes a sensitive radioactive assay for PDE activity in which substrate and product can be quickly and easily separated by thin-layer chromatography.

Topics: Chromatography, Thin Layer; Cyclic GMP; Enzyme Activation; Enzyme Assays; Phosphoric Diester Hydrolases; Phosphorus Radioisotopes

Bacterial signaling networks control a wide variety of cellular processes including growth, metabolism, and pathogenesis. Bis-(3'-5')-cyclic dimeric guanosine monophosphate (cdiGMP) is a secondary signaling nucleotide that controls cellulose synthesis, biofilm formation, motility and virulence in a wide range of gram-negative bacterial species. CdiGMP is a dynamic molecule that forms different tertiary structures in vitro, including a trans-monomer, cis-monomer, cis-dimer and G-octaplex (G8). Although the monomer and dimer have been shown to be physiologically relevant in modulating protein activity and transcription, the biological effects of the cdiGMP G8 has not yet been described. Here, we have developed a TLC-based assay to detect radiolabeled cdiGMP G8 formation. Utilizing the radiolabeled cdiGMP G8, we have also shown a novel inhibitory interaction between the cdiGMP G8 and HIV-1 reverse transcriptase and that the cdiGMP G8 does not interact with proteins from Pseudomonas aeruginosa known to bind monomeric and dimeric cdiGMP. These results suggest that the radiolabeled cdiGMP G8 can be used to measure interactions between the cdiGMP G8 and cellular proteins, providing an avenue through which the biological significance of this molecule could be investigated.

Topics: Bacterial Proteins; Chromatography, Thin Layer; Cyclic GMP; HIV Reverse Transcriptase; HIV-1; Phosphorus Radioisotopes; Polymerization; Protein Binding; Protein Structure, Tertiary; Pseudomonas aeruginosa; Signal Transduction; Solutions; Staining and Labeling; Stereoisomerism

The occurrence of the novel regulatory nucleotide bis(3',5')-cyclic diguanylic acid (c-di-GMP) and its relation to cellulose biogenesis in the plant pathogen Agrobacterium tumefaciens was studied. c-di-GMP was detected in acid extracts of 32P-labeled cells grown in various media, and an enzyme responsible for its formation from GTP was found to be present in cell-free preparations. Cellulose synthesis in vivo was quantitatively assessed with [14C]glucose as a tracer. The organism produced cellulose during growth in the absence of plant cells, and this capacity was retained in resting cells. Synthesis of a cellulosic product from UDP-glucose in vitro with membrane preparations was markedly stimulated by c-di-GMP and its precursor GTP and was further enhanced by Ca2+. The calcium effect was attributed to inhibition of a c-di-GMP-degrading enzyme shown to be present in the cellulose synthase-containing membranes.

Topics: Arabidopsis Proteins; Cellulose; Cyclic GMP; Glucosyltransferases; Guanosine Triphosphate; Kinetics; Phosphorus Radioisotopes; Radioisotope Dilution Technique; Rhizobium