During its life cycle Caulobacter crescentus divides asymmetrically to produce a motile, flagellated swarmer (SW) and a sessile, surface adherent stalked (ST) cell. Whereas the ST cell progeny immediately enters a new round of cell division, the SW cell first differentiates into a ST cell before it initiates DNA replication and cell division. Cell polarity and cell cycle progression are implemented by oscillating global transcriptional regulators and by spatially dynamic phosphosignaling and proteolysis pathways. Recent studies have identified the second messenger c-di-GMP as an integral part of this regulatory network and have indicated that c-di-GMP not only controls the motile-sessile switch, but is also a key component of C. crescentus cell cycle progression. However, many of the c-di-GMP network components involved in these reguatory processes are still waiting to be identified or have not been studied in detail.
C-di-GMP control modules consist of diguanylate cyclases (c-di-GMP synthesis), phosphodiesterases (c-di-GMP degradation), and specific effector proteins that mediate a cellular response through the c-di-GMP dependent interaction with downstream target molecules. The C. crescentus chromosome encodes a total of 14 potential diguanylate cyclases (GGDEF domain proteins) or phosphodiesterases (EAL domain proteins), only a fraction of which has been analyzed so far. Preliminary mutational analyses of all GGDEF/EAL domain proteins revealed that c-di-GMP regulates all aspects of C. crescentus pole development and have indicated that most of these components are involved in cell polarity control. Based on these observations we propose to embark on a global analysis of the c-di-GMP network that operates in Caulobacter crescentus to coordinate cell cycle progression and pole morphogenesis. In the first part (subproject A) we propose an in-depth analysis of a tripartite c-di-GMP control module responsible for S-phase entry and the motile-sessile transition. As these components are tightly regulated in time and space during the cell cycle, they provide a paradigm to investigate the spatial and temporal regulatory mechanisms operating in c-di-GMP modules. In the second part (subproject B), we propose to expand these studies to all other c-di-GMP signaling components present in C. crescentus and to analyze their molecular and cellular functions, their interaction partners, and their spatio-temporal control. We will make use of a strain devoid of c-di-GMP to globally analyze transcription, translation, and protein levels in dependency of c-di-GMP. These experiments will begin to shed light on the global cellular network that controls C. crescentus growth and behavior through the controlled production of c-di-GMP. In the thrid part (subproject C) we propose to develop and use new tools to biochemically identify novel c-di-GMP effector proteins and their downstream targets. This will complement the c-di-GMP control modules by adding the much sought-after effector molecules, and will provide an entry point into the identification of downstream targets and link them to c-di-GMP dependent cellular processes. Finally, in part 4 (subproject D) we plan to use sophisticated live cell assays to screen a chemical library for small molecule inhibitors of diguanylate cyclases, the key enzymes of the c-di-GMP signaling network. This will provide valuable compounds for a chemical genetics approach to analyze c-di-GMP signaling in C. crescentus and in a range of pathogenic bacteria that display a c-di-GMP signaling network of much higher complexity. These studies will provide an in-depth view on c-di-GMP mediated control at the molecular and cellular level and will generate a global and quantitative understanding of the c-di-GMP signaling network in the C. crescentus model organism. This will lay the foundation for future modeling and engineering approaches to approximate the temporal, spatial and dynamic properties of the regulatory system.