The PAMP c-di-AMP Is Essential for Listeria monocytogenes Growth in Rich but Not Minimal Media due to a Toxic Increase in (p)ppGpp

Cyclic di-adenosine monophosphate (c-di-AMP) is a widely distributed second messenger that appears to be essential in multiple bacterial species, including the Gram-positive facultative intracellular pathogen Listeria monocytogenes. In this study, the only L. monocytogenes diadenylate cyclase gene, dacA, was deleted using a Cre-lox system activated during infection of cultured macrophages. All ΔdacA strains recovered from infected cells harbored one or more suppressor mutations that allowed growth in the absence of c-di-AMP. Suppressor mutations in the synthase domain of the bi-functional (p)ppGpp synthase/hydrolase led to reduced (p)ppGpp levels. A genetic assay confirmed that dacA was essential in wild-type but not strains lacking all three (p)ppGpp synthases. Further genetic analysis suggested that c-di-AMP was essential because accumulated (p)ppGpp altered GTP concentrations, thereby inactivating the pleiotropic transcriptional regulator CodY. We propose that c-di-AMP is conditionally essential for metabolic changes that occur in growth in rich medium and host cells but not minimal medium. [Display omitted] •c-di-AMP is predicted to be essential in many pathogens and is thus an ideal PAMP•ΔdacA mutants lacking c-di-AMP contain suppressor mutations within a (p)ppGpp synthase•Decreased c-di-AMP increases (p)ppGpp, which inhibits growth via inactivation of CodY•In minimal medium, which favors CodY inactivation, dacA is no longer essential c-di-AMP is a nucleotide second messenger recognized by the innate immune system and is predicted to be essential in many bacteria. Whiteley et al. show that depletion of c-di-AMP within Listeria monocytogenes leads to accumulation of (p)ppGpp, which inhibits bacterial growth but is also necessary for virulence.