SummaryBiofilms are communities of surface-attached, matrixembedded 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-b-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 signalling molecules guanosine-bis 3Ј, 5Ј(diphosphate) (ppGpp) and bis-(3Ј-5Ј)-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 signalling 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 signalling might represent an effective means for biofilm control during chronic infections.

Cyclic diguanosine-monophosphate (c-di-GMP) is a bacterial signaling molecule that triggers a switch from motile to sessile bacterial lifestyles. This mechanism is of considerable pharmaceutical interest, since it is related to bacterial virulence, biofilm formation, and persistence of infection. Previously, c-di-GMP has been reported to display a rich polymorphism of various oligomeric forms at millimolar concentrations, which differ in base stacking and G-quartet interactions. Here, we have analyzed the equilibrium and exchange kinetics between these various forms by NMR spectroscopy. We find that the association of the monomer into a dimeric form is in fast exchange (

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β-barrel proteins are key functional components of the outer membranes of gram-negative bacteria, mitochondria and plastids. They mediate transport across the membrane, act as receptors and are involved in bacterial pathogenicity. Despite their crucial roles, assembly and membrane insertion of β-barrel outer membrane proteins, which are mediated by β-barrel membrane proteins of the OMP85 family, have remained elusive. The crystal structure of the Escherichia coli OMP85 protein TamA [1], which is involved in autotransporter biogenesis, now provides a novel perspective on β-barrel membrane protein assembly. The protein was crystallized in lipidic phase and microseeding was employed to obtain high-quality 2.3 Å diffraction data. TamA comprises a 16-stranded transmembrane β-barrel and three N-terminal POTRA domains. The barrel is closed at the extracellular face by a conserved lid loop tightly interacting with a conserved lock region on the inner barrel wall. The C-terminal β-strand of the barrel forms an unusual inward kink, which creates a gate for substrate access to the lipid bilayer and weakens lateral inter-strand connection. These structural features immediately suggest a mechanism of autotransporter insertion based on barrel expansion and lateral release. Based on structural conservation of all core elements [2], this mechanism might well be relevant for the entire OMP85 family.[1] Gruss F, Zähringer F, Jakob RP, et al., The structural basis of autotransporter translocation by TamA. Nat Struct Mol Biol. (2013) 20:1318

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