Thesis (Ph.D.)--University of Washington, 2012c-di-GMP is a second messenger that regulates motility and the production of adhesive factors in many bacterial species. Enzymes containing specific c-di-GMP metabolizing domains integrate information about the environment into an intracellular level of c-di-GMP that then binds to specific downstream receptors, including proteins that contain the PilZ domain. Many bacterial species encode dozens of c-di-GMP metabolizing enzymes in their genomes. Although each of these enzymes metabolizes the same small, diffusible second messenger molecule, many of these proteins can be specifically linked to downstream c-di-GMP-regulated processes. The mechanisms involved in achieving this signaling specificity between c-di-GMP metabolizing enzymes and their downstream receptors are not known. Here, we provide evidence that c-di-GMP signaling specificity is achieved through differences in the binding affinities of downstream receptors. Salmonella Typhimurium harbors two PilZ domain proteins: YcgR, which controls flagellar-based motility, and BcsA, an enzyme that produces cellulose. Using a Forster resonance energy transfer (FRET)-based method, we measured the binding affinities of these PilZ domain proteins and found that they span a 43-fold range. Increasing the binding affinity of BcsA for c-di-GMP increased the amount of cellulose that this enzyme produced at lower levels of c-di-GMP. Decreasing the affinity of YcgR for c-di-GMP increased the amount of this second messenger needed for YcgR to inhibit motility. In addition, we found that mutation in yhjH, which encodes a predicted c-di-GMP-degrading enzyme, increased the fraction of the cellular population that demonstrated c-di-GMP levels high enough to bind to the higher-affinity YcgR protein, but did not enough to bind to the lower-affinity BcsA protein and stimulate cellulose production. Thus, the specific affinities of these proteins for c-di-GMP are important for their biological functions. Additionally, the binding affinities of the eight PilZ domain proteins in Pseudomonas aeruginosa were measured and found to span a 145-fold range, implying that regulation by binding affinity of downstream receptors for c-di-GMP may be a common theme in c-di-GMP signaling. Finally, we generated a panel of FRET-based c-di-GMP biosensors which will allow for the accurate measurement of the free c-di-GMP level in individual cells from the nanomolar to the micromolar range
