Our primary goal is to investigate the molecular details of signaling via the second messenger c-dl-GMP. It is clear that this molecule binds to a series of receptors that regulate function as adaptors, modifying downstream functions. One of the key discoveries in the field of c-di-GMP regulation by others and us has been the range of proteins that function as receptors for this regulatory dinucleotide. This includes the discovery in 2008 by Breaker's group in V. cholerae of a c-di-GMP binding riboswitch, Riboswitches are structured RNAs at 5' ends of transcripts that regulate translation following interaction with a ligand. The total number and diversity of c-di-GMP receptors in any given organism is therefore unclear and little Is known about the regulatory activities of these adapter molecules. A wealth of tools is now available to Investigate the c-di-GMP signaling pathway: (I) two complete ordered libraries of transposon insertions P. aerug/nosa strains PA01 and PAU. (ii) c-dl-GMP modified with biotin that provides an affinity purification reagent for c-di-GMP binding proteins and RNAs (iii) a validated bacterial bioassay to detect interaction between c-di-GMP and its receptors. We will use these and more standard genetic and biochemical tools to accomplish the following objectives over the next five years. Carry out a proteome-wide screen for receptors of c-di-GMP. Follow this up by a two-hybrid library screen for proteins that Interact with these c-di-GMP-binding proteins. Following identification the proteins by mass spectrometry, the corresponding mutants (retrieved from the library of P. aeruginosa transposon insertions) will be tested for a variety of phenotypes based on sequence analysis of the genes, Carry out a genome-wide screen for RNA c-di-GMP receptors (riboswitches). To this end, we will adapt the genomic SELEX method to identify all potential RNA sequences that Interact with c-di-GMP. Presumably, c-di-GMP controls the translation and/or stability of transcripts and this will be verified though engineered mutations and an assessment of c-di-GMP dependent synthesis of the protein products. Determine the crystal structures of all unique families of c-di-GMP-binding proteins complexed with their ligands to determine the structural basis of recognition of this signaling molecule. Extensive mutagenesis study will be undertaken to define the contact residues responsible for substrate recognition, Determine the sub-cellular localization of the c-dl-GMP receptors an their putative interactive partners. Various single-cell techniques Including fluorescence microscopy, use of split-GFP constructs will be employed lo demonstrate interaction of the various components of the signaling complexes at specific locations In the cells. Determine the molecular basis of regulation of expression of enzymes (DGCs and PDEs) and c-di-GMP binding proteins and RNAs, In addition to following up on previously identified RsmA, RsmZ and RsmY regulatory pathway, we will carry out a large scale transposon mutagenesis screen (using appropriate reporter gene fusions) to identify additional regulatory factors that control expression of the components of the c-di-GMP signaling pathways. The continuation of this project should provided new information about a broadly-conserved system of signal transduction, with the P, aeruginosa systems serving as a prototype for understanding the activities analogues signaling pathways that control the expression of virulence factors in a significant numberof bacterial pathogens.