Shieh, A. | bioRxiv Science

bioRxiv2026

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.

microbiology↗

bioRxiv2019

CpG-creating Mutations are Costly in Many Human Viruses

Mutations can occur throughout the virus genome and may be beneficial or deleterious. We are interested in mutations that yield a C next to a G, producing CpG sites. CpG sites are rare in eukaryotic and viral genomes. For the eukaryotes, it is thought that CpG sites are rare because they are prone to mutation when methylated. In viruses, we know less about why CpG sites are rare. A previous study in HIV suggested that CpG-creating transition mutations are more costly that similar non-CpG-creating mutations. To determine if this is the case in other viruses, we analyzed the allele frequencies of CpG-creating and non-CpG-creating mutations across various strains, subtypes, and genes of viruses using existing data obtained from Genbank, HIV Databases, and Virus Pathogen Resource. Our results suggest that CpG sites are costly for most viruses. By understanding the cost of CpG sites, we can obtain further insights into the evolution and adaptation of viruses.

evolutionary biology↗