Second messenger signaling governs Escherichia coli biofilm induction upon ribosomal stress

Biofilms are communities of surface attached, matrix embedded microbial cells that can resist antimicrobial chemotherapy and contribute to persistent infections. Using an Escherichia coli biofilm model we found that exposure of bacteria to subinhibitory concentrations of ribosome-targeting antibiotics leads to strong biofilm induction. We present evidence that this effect is elicited by the ribosome in response to translational stress. Biofilm induction involves upregulation of the polysaccharide adhesin poly-beta-1,6-N-acetyl-glucosamine (poly-GlcNAc) and two components of the poly-GlcNAc biosynthesis machinery, PgaA and PgaD. Poly-GlcNAc control depends on the bacterial signaling molecules guanosine-bis 3', 5'(diphosphate) (ppGpp) and bis-(3'-5')-cyclic di-GMP (c-di-GMP). Treatment with translation inhibitors causes a ppGpp-hydrolase (SpoT)-mediated reduction of ppGpp levels, resulting in specific derepression of PgaA. Maximal induction of PgaD and poly-GlcNAc synthesis requires the production of c-di-GMP by the dedicated diguanylate cyclase YdeH. Our results identify a novel regulatory mechanism that relies on ppGpp signaling to relay information about ribosomal performance to the Pga machinery, thereby inducing adhesin production and biofilm formation. Based on the important synergistic roles of ppGpp and c-di-GMP in this process, we suggest that interference with bacterial second messenger signaling might represent an effective means for biofilm control during chronic infections.