title: c-di-AMP modulates Listeria monocytogenes central metabolism to regulate growth, antibiotic resistance and osmoregulation.
abstract: 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 ΔdacA mutants are highly sensitive to the β-lactam antibiotic cefuroxime. In this study, loss of function mutations in the oligopeptide importer (oppABCDF) and glycine betaine importer (gbuABC) allowed ΔdacA mutants to grow in rich medium. Since oligopeptides were sufficient to inhibit growth of the ΔdacA mutant we hypothesized that oligopeptides act as osmolytes, similar to glycine betaine, to disrupt intracellular osmotic pressure. Supplementation with salt stabilized the ΔdacA 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 ΔdacA 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 ΔdacA phenotypes. These data suggested that c-di-AMP modulates central metabolism at the pyruvate node to moderate citrate production and indeed, the ΔdacA mutant accumulated six times the concentration of citrate present in wild-type bacteria.
authors.affiliation: Graduate Group in Infectious Diseases and Immunity, School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
authors.forename: Aaron T
authors.initials: AT
authors.lastname: Whiteley
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
authors.forename: Nicholas E
authors.initials: NE
authors.lastname: Garelis
authors.affiliation: Graduate Group in Microbiology, University of California, Berkeley, Berkeley, CA, USA.
authors.forename: Bret N
authors.initials: BN
authors.lastname: Peterson
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY, USA.
authors.forename: Philip H
authors.initials: PH
authors.lastname: Choi
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY, USA.
authors.forename: Liang
authors.initials: L
authors.lastname: Tong
authors.affiliation: Department of Microbiology, University of Washington, Seattle, WA, USA.
authors.forename: Joshua J
authors.initials: JJ
authors.lastname: Woodward
authors.affiliation: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.|School of Public Health, University of California, Berkeley, Berkeley, CA, USA.
authors.forename: Daniel A
authors.identifier: 0000-0003-1218-2799
authors.initials: DA
authors.lastname: Portnoy
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism; D002473:Cell Wall / Q000378:metabolism; D015226:Dinucleoside Phosphates / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D004352:Drug Resistance, Microbial; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D008089:Listeria monocytogenes / Q000254:growth & development / Q000378:metabolism*; D064587:Osmoregulation / Q000502:physiology; D009997:Osmotic Pressure; D019761:Phosphorus-Oxygen Lyases / Q000378:metabolism; D011766:Pyruvate Carboxylase / Q000378:metabolism; D015290:Second Messenger Systems; D013489:Suppression, Genetic
chemical_list: D001426:Bacterial Proteins; D015226:Dinucleoside Phosphates; C528998:cyclic diadenosine phosphate; D000105:Acetyl Coenzyme A; D019761:Phosphorus-Oxygen Lyases; D011766:Pyruvate Carboxylase