Analysis of Bacterial-Mediated c-di-AMP Degradation by Thin-Layer Chromatography

Cyclic di-AMP is a bacterial second messenger nucleotide required for the regulation of numerous cellular functions, including potassium and osmolyte homeostasis, DNA repair, cell wall integrity, central metabolism, and stress adaptation. This second messenger is synthesized from two ATP molecules by diadenylate cyclases (DAC) and degraded by cytoplasmic and surface-associated phosphodiesterases (PDE) to phosphoadenylyl adenosine (5' pApA), adenosine monophosphate (AMP), and, in some instances, adenosine and inorganic phosphate (Pi). Levels of c-di-AMP in bacteria can be determined using different methods, including liquid chromatography-mass spectrometry (LC-MS/MS), enzyme-linked immunosorbent assay (ELISA), and luminescent and fluorescent biosensors. Thin-layer chromatography (TLC) is another method routinely used to monitor c-di-AMP synthesis and degradation by purified DAC and PDE enzymes and is particularly useful for monitoring c-di-AMP degradation products. Here, we devised a TLC-based method to monitor extracellular c-di-AMP stability and degradation by intact bacterial cells using radiolabeled c-di-AMP. We show that bacterial strains of Enterococcus faecalis and Streptococcus agalactiae that possess surface-associated PDEs can rapidly degrade extracellular c-di-AMP. In addition, we demonstrate that this method can be used to indirectly identify alternative enzyme substrates through competition assays. We propose that this TLC-based assay is an efficient method to analyze bacterial-mediated degradation of c-di-AMP and is amenable to testing other radiolabeled nucleotides. Key features • This method makes use of TLC, a well-established technique, to visualize c-di-AMP degradation products. • Bypasses the need to obtain purified protein and instead uses live bacterial cells to monitor extracellular substrate degradation at the cell surface interface. • This protocol can be adapted to other organisms and cell types and to detect other radiolabeled substrates.