Key Molecular Mechanisms of c-di-GMP Signaling in Bacterial Biofilm Formation

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Bacterial biofilms colonize diverse surfaces and are highly resistant against antibiotics or the immune system. In these biofilms, cells are embedded in a self-produced matrix consisting of secreted proteins, amyloid fibres (e.g. curli), exopolysaccharides (e.g. cellulose) and even DNA. These matrix components determine the complex microarchitecture and morphology of these biofilms. In E. coli, synthesis of curli fibres and cellulose is under control of the stationary phase sigma factor RpoS and the second messenger c-di-GMP. The latter is produced by diguanylate cyclases (DGC, with GGDEF domains) and is degraded by specific phosphodiesterases (PDE, with EAL domains). Many of these differentially expressed DGCs and PDEs are membrane-associated and activity-controlled via N-terminal sensory domains. Many bacteria possess multiple GGDEF/EAL domain proteins (29 in E. coli K-12), which has led to the concept of locally acting c-di-GMP control modules based on direct and highly specific protein-protein interactions. Our recent analysis of YciR, the first and paradigmatic trigger enzyme in bacterial second messenger signaling and a key switching device for the production of curli and cellulose, has demonstrated how this can work: YciR is at the same time (i) a regulator that inhibits two target proteins by direct interaction, (ii) a PDE, and (iii) a c-di-GMP effector component, because its binding and degradation of c-di-GMP releases its direct interaction with the target proteins (a DGC and a transcription factor required to produce curli and cellulose).The project proposed here will focus on the molecular mechanisms that underly two key aspects of c-di-GMP signaling:1. Local c-di-GMP signaling that involves specific protein-protein interactions of c-di-GMP-related trigger enzymes: here, the potential role of YciR acting also in conjunction with three additional DGCs, as well as the molecular functions of two additional PDEs and putative trigger enzymes that are involved in cellulose biosynthesis and direct regulation of gene expression, respectively, will be characterized.2. Sensory input into c-di-GMP signaling: here, the goal will be to identify novel environmental input signals into biofilm formation and to assign specific signal perception to distinct DGCs and PDEs. The molecular mechanisms of signal processing by specific N-terminal sensory domains of DGCs and PDEs will be characterized with a particular focus on redox-responsive PDEs and multiple signal integration by multi-domain DGCs and PDEs.Finally, the integration of these molecular mechanisms within the large regulatory networks that generate functionality of a biofilm will be elucidated. Since c-di-GMP signaling is almost ubiquitously used by bacteria to control biofilm formation, insights generated by this project will open new perspectives on anti-biofilm strategies and drugs.