Synthesis of [32P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations

Methods Mol Biol. 2017:1657:23-29. doi: 10.1007/978-1-4939-7240-1_3.

Abstract

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.

Keywords: Cyclic-di-GMP; Diguanylate cyclase; Phosphodiesterase.

MeSH terms

  • 3',5'-Cyclic-GMP Phosphodiesterases / genetics
  • 3',5'-Cyclic-GMP Phosphodiesterases / isolation & purification
  • 3',5'-Cyclic-GMP Phosphodiesterases / metabolism*
  • Bacteria / genetics
  • Bacteria / metabolism
  • Cyclic GMP / analogs & derivatives*
  • Cyclic GMP / chemical synthesis
  • Enzyme Activation
  • Escherichia coli Proteins / genetics
  • Escherichia coli Proteins / isolation & purification
  • Escherichia coli Proteins / metabolism*
  • Gene Expression
  • Phosphorus Radioisotopes* / chemistry
  • Phosphorus-Oxygen Lyases / genetics
  • Phosphorus-Oxygen Lyases / isolation & purification
  • Phosphorus-Oxygen Lyases / metabolism*
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism

Substances

  • Escherichia coli Proteins
  • Phosphorus Radioisotopes
  • Recombinant Proteins
  • bis(3',5')-cyclic diguanylic acid
  • Phosphorus-32
  • 3',5'-Cyclic-GMP Phosphodiesterases
  • Phosphorus-Oxygen Lyases
  • diguanylate cyclase
  • Cyclic GMP