Microorganisms have diverse survival strategies for increasing their fitness in response tochallenging environmental conditions. Many of these survival strategies require signal sensing systems to relay environmental input to cellular output. One of the ubiquitous signaling systems used by microorganisms is c-di-GMP signaling. C-di-GMP is produced and degraded by diguanylate cyclases (DGCs) and phosphodiesterases (PDEs), respectively, and many of these proteins contain signal-sensing domains to modulate their enzymatic activity. C-di-GMP signaling regulates key bacterial processes such as biofilm formation, motility, and virulence in response to varying environmental parameters. Here I first review recent successful strategies for discovering and developing c-di-GMPspecific inhibitors with the goal of acting as therapeutics for the prevention and dispersal of biofilms. Next, I discuss the results of my studies designed to understand the transcriptional regulation of a key DGC, VpvC. I found that the vpvABC system is under the control of a c-di- GMP sensing circuit and a stationary growth phase circuit, which together are responsible for the transcription of vpvABC. Finally, I discuss my studies investigating the V. cholerae RpoS regulon in biofilm-grown cells with high and low c-di-GMP cellular c-di-GMP levels. In biofilmgrown cells, RpoS regulates stress response, chemotaxis and motility, biofilm formation, and pathogenesis; some of these processes are regulated in a cellular c-di-GMP level-dependent manner. Additionally, I discovered that RpoS positively regulates the transcription of a sizable number of genes encoding DGCs and PDEs, demonstrating RpoS's significance in c-di-GMP signaling specificity in V. cholerae.