A metabolic checkpoint coordinates bacterial cell envelope biosynthesis and c-di-GMP signaling

The bacterial second messenger cyclic dimeric guanosine monophosphate (c-di-GMP) drives the transition from motile to biofilm lifestyles, yet the mechanisms by which bacteria couple cell envelope stress to c-di-GMP signaling remain poorly understood. Here, we show that sub-inhibitory concentrations of antibiotics targeting early cytoplasmic steps of peptidoglycan (PG) biosynthesis, but not inhibitors of PG polymerization, membrane integrity, or other intracellular processes, specifically elevate intracellular c-di-GMP levels in Pseudomonas aeruginosa . Using live-cell imaging and in vitro enzymatic assays, we demonstrate that this elevation results from reduced phosphodiesterase (PDE) activity rather than increased diguanylate cyclase (DGC) activity, with multiple PDEs contributing to the response. A screen of the complete P. aeruginosa DGC/PDE mutant library identified DipA, BifA, RocR, and RmcA as the primary PDEs mediating this effect. Strikingly, we find that acetyl-CoA, a central metabolite consumed during cell envelope precursor biosynthesis, directly inhibits PDE activity by competing for the conserved EAL domain active site, as supported by biochemical assays with purified RocR and molecular docking analysis. Because EAL domain residues that contact acetyl-CoA are broadly conserved across bacterial species, this mechanism may represent a widespread strategy for sensing metabolic perturbations of cell envelope synthesis. Together, these findings reveal that P. aeruginosa monitors the metabolic status of cell envelope biogenesis through acetyl-CoA-mediated allosteric inhibition of c-di-GMP PDEs, linking envelope biosynthetic flux to adaptive biofilm formation.