Spatiotemporal control of c-di-GMP signaling in Shewanella via phosphodiesterase PdeB

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Bacterial cells possess a high degree of intracellular organization, which require appropriate spatiotemporal regulational patterns. c-di-GMP has emerged as important intracellular second messenger molecule which is involved in regulation of a wide array of processes in numerous bacterial species. Most bacterial species harbor a plethora of proteins involved in c-di-GMP metabolism and various potential c-d-GMP-binding proteins, raising the question of how the appropriate spatiotemporal regulatory responses are elicited. In previous studies we have explored mechanisms by which gammaproteobacteria of the genus Shewanella recruit the flagellar and chemotaxis systems to the appropriate cell pole. Recently, we have identified a c-di-GMP-degrading phosphodiesterase PdeB which is crucial for proper function of the flagellar motor. PdeB is recruited to the flagellated cell pole in dependence of the polar marker HubP. Our preliminary data strongly indicate that PdeB affects the performance of the flagellar motor by an unknown mechanism, putatively upon perceiving a signal via its periplasmic domain with an unknown structure. Within this proposed project, we will take efforts to elucidate the signal input as well as the mechanism by which PdeB affects the flagellar motor, and we are aiming at understanding how spatiotemporal localization of a phosphodiesterase specifically affects cellular processes. To this end, we will heterologously produce and purify domains of PdeB to determine their activity, the hitherto uncharacterized putative signal-perceiving domain will be crystallized to identify structure and potential mechanism of signal perception. Protein pull-downs and interaction studies will identify potential factors mediating polar recruitment, signal perception, and phenotypic output of PdeB. To determine the mechanism by which c-di-GMP levels affect flagellar functions, we will perform direct binding studies with major components of the flagella and chemotaxis systems. In parallel, we will use a global mutagenesis approach and a c-di-GMP-binding protein capture assay to unveil factors of the c-di-GMP regulatory network with respect to motility and further cellular processes. By fluorescence microscopy approaches, the localization patterns of PdeB will be determined. In a complementary approach we will develop a genetic system that will allow targeting of c-d-GMP-degrading or -synthesizing activity to specific cell compartments. This system will be applied to specifically study the effect of local activity on the distribution of c-di-GMP and regulation of cellular processes.