![]() These findings establish CDA-Luc as a more affordable and sensitive alternative to conventional c-di-AMP detection tools with broad utility for the study of bacterial cyclic dinucleotide physiology.Ĭyclic dinucleotide (CDN) second messengers have emerged as conserved signaling molecules across all domains of life. We also demonstrate the utility of this approach for the quantification of the cyclic oligonucleotide-based anti-phage signaling system (CBASS) effector, 3’3’-cGAMP. To address this limitation, we describe here the development of a luciferase-based coupled enzyme assay that leverages the cyclic nucleotide phosphodiesterase, CnpB, for the sensitive and high throughput quantification of 3’3’-c-di-AMP. Despite significant advancements in the field, there is still a major gap in the understanding of the environmental and cellular factors that influence c-di-AMP dynamics due to a lack of tools to sensitively and rapidly monitor changes in c-di-AMP levels. We and others have shown that defects in c-di-AMP homeostasis result in severe physiological defects and virulence attenuation in many bacterial species. In recent years, the secondary messenger, cyclic di-(3’,5’)-adenosine monophosphate (c-di-AMP), has been identified as an essential signaling molecule in a diverse array of bacterial genera. Cyclic dinucleotide signaling systems are found ubiquitously throughout nature allowing organisms to rapidly and dynamically sense and respond to alterations in their environments.
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