Sensory Mechanisms and Local Signaling in c-di-GMP-mediated Signal Transduction in Escherichia coli

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Bacterial biofilms colonize diverse surfaces and are highly resistant against antibiotics or host immune systems. In these biofilms, cells are embedded in a self-produced protective matrix of extracellular biopolymers. Major biofilm matrix components in E. coli are amyloid curli fibres and the exopolysaccharide cellulose whose synthesis is under control of the stationary phase sigma factor RpoS (¿S) 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). 25 of the 29 GGDEF/EAL domain proteins of E. coli K-12 are enzymatically active, with 12 being DGCs and 13 being PDEs. Many of these differentially expressed DGCs and PDEs are membrane-associated and their activities are controlled by N-terminal sensory domains.Using biofilm matrix production as a c-di-GMP target that provides for convenient phenotypes and readouts for genetic, molecular biological and biochemical analyses, the proposed project aims at clarifying the molecular mechanisms of signal sensing and processing at the primary and secondary levels of c-di-GMP signaling in E. coli. This includes (i) sensing environmental and cellular signals via the N-terminal sensory input domains of a novel class of PDEs, and (ii) sensing c-di-GMP via novel and unorthodox c-di-GMP effector mechanisms. In particular, the following signal perception and c-di-GMP effector mechanisms will be studied:- Redox control of cytoplasmic enzyme activity via disulfide bond formation and proteolytic processing in the periplasmic loop domain of the six E. coli PDEs with CSS-EAL domain architecture- Molecular structure, function and targets of YbjK, a DeoR-like transcription factor that seems to bind c-di-GMP as a G-quadruplex- Regulation of transcription by PdeL, a locally acting c-di-GMP-binding and ¿degrading 'trigger PDE' and transcription factor with a LuxR-EAL domain architecture- Structure and function of a putative c-di-GMP riboswitch in the intergenic region of the mRNA of the orf0317-pdeL operon, which down-regulates PdeL expression.Furthermore, the integration of these molecular processes within the large regulatory network that controls matrix production and thereby generates functionality and architecture of a biofilm will be elucidated.With its focus on sensory input in the control of DGCs and PDEs, c-di-GMP-sensing effector components and local c-di-GMP signaling, this project is expected to make a major contribution to achieving the goals of SPP 1879. Moreover, its results will have practical applications, e.g. in the development of novel anti-biofilm strategies or in synthetic biology.