c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation

Cyclic diadenosine monophosphate (c-di-AMP) is a conserved nucleotide second messenger critical for bacterial growth and resistance to cell wall-active antibiotics. In Listeria monocytogenes, the sole diadenylate cyclase, DacA, is essential in rich, but not synthetic media and dacA mutants are highly sensitive to the -lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed dacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the dacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the dacA mutant in rich medium and restored cefuroxime resistance. Additional suppressor mutations in the acetyl-CoA binding site of pyruvate carboxylase (PycA) rescued cefuroxime resistance and resulted in a 100-fold increase in virulence of the dacA mutant. PycA is inhibited by c-di-AMP and these mutations prompted us to examine the role of TCA cycle enzymes. Inactivation of citrate synthase, but not down-stream enzymes suppressed dacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the dacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.