Sensory Mechanisms and Local Signaling in C-Di-GMP-Mediated Signal Transduction in Escherichia coli

Signal transduction via secondary messengers involves primary and secondary signal perception, since both primary conditions—e.g., certain molecules, oxygen, redox signals, light, or the state of a partner protein—trigger the synthesis or degradation of the secondary messenger, and the secondary messenger itself must also be perceived in order to control the “output” of a signaling pathway. The aim of the project is to characterize the molecular mechanisms of both primary and secondary signal perception in the c-di-GMP-mediated key signaling pathways in E. coli. This bacterium has 12 diguanylate cyclases (DGCs) and 13 c-di-GMP-specific phosphodiesterases (PDEs), many of which are involved in a fundamental c-di-GMP signaling pathway: the production of amyloid curli fibers and phosphoethanolamine-modified (pEtN) cellulose as polymeric components of the extracellular biofilm matrix. Local c-di-GMP-mediated signal transduction also occurs prominently in E. coli, based on the formation of complexes of DGCs, PDEs, and effector/target systems, through which c-di-GMP signaling pathways can operate in parallel without altering the cellular c-di-GMP concentration, which is kept very low by a highly expressed master PDE. The project consists of three parts:
- Periplasmic redox signal processing in CSS domain PDEs: Comparative structural and functional analyses will clarify how a conserved disulfide/thiol switch in the periplasmic CSS domains of the five PDEs of this type in E. coli controls the PDE activity of the cytoplasmic EAL domains.
- The DgcE-CsgD signal transduction pathway in the control of matrix components: The goal here is to elucidate all essential steps from primary signal input (via a GTPase interacting with DgcE) via local c-di-GMP signal transduction (via the DgcE/PdeR/ DgcM/MlrA signaling pathway) to a novel c-di-GMP-dependent transcription initiation mechanism (at the promoter of csgD, which encodes the central activator of curli and pEtN cellulose synthesis).
- System-wide molecular analysis of local c-di-GMP perception and signal transduction in E. coli: Here, known and new c-di-GMP effector proteins will be systematically investigated for their potential direct interactions with all 12 DGCs. A new riboswitch-based fluorescent probe will be used to determine c-di-GMP concentrations in situ in different biofilm zones. Finally, mathematical modeling of local c-di-GMP signal transduction in non-compartmentalized systems will be performed.

By focusing on sensory input in the control of DGCs and PDEs, c-di-GMP-binding effector components, and mechanisms of local c-di-GMP signal transduction, this project will contribute substantially to the achievement of the objectives of SPP 1879.