Abstract The majority of chronic bacterial infections have been attributed to biofilm formation. Biofilm formation is a conserved physiological process during which bacteria become sessile, secrete a protective extracellular matrix and function as a community, rather than as single cells. It is an adaptation mechanism, which starts with environmental cues that are transduced via cell signaling pathways and ultimately translated into changes in cellular behavior. The dinucleotide second messenger c-di-GMP, together with the enzymes for its production and degradation, has been identified as the major intracellular signaling molecule that controls biofilm formation and virulence in many bacterial species. Many microbes encode a large number of enzymes involved in c-di-GMP metabolism and receptors for c-di-GMP-dependent responses, and this number often scales with the adaptation potential of the organism. The prevalence and organization of c-di-GMP signaling networks suggests that mechanisms exist to ensure signaling specificity, although this hypothesis has not been explored in great detail. Here, studies will focus on the regulation of a conserved signaling network that controls cell adhesion in a wide range of bacteria, including several major human pathogens. Central to this regulatory node is a transmembrane c-di-GMP receptor with a prevalent domain organization and the enzymes that control its activity. Preliminary data indicate that this system is ideal to study a major open question in the field: How is c-di-GMP signaling specificity achieved in signaling networks containing dozens of proteins with identical catalytic activities? We address this question several ways by focusing on the conserved, membrane-bound, HAMP domain-containing c-di-GMP receptor LapD. We explore how protein- protein interactions between this receptor and c-di-GMP metabolizing enzymes help confer specificity. Through these studies we also address how c-di-GMP signaling is controlled across the cell membrane, and how this protein family, comprised of >2000 HAMP-GGDEF-EAL domain-containing proteins, is regulated. Finally, we utilize mass spectroscopy-based proteomics approaches to systematically identify c-di-GMP- relevant protein networks. These studies will be complemented by the elucidation of specific responses and signaling networks responsive to physiological inputs, foremost nutritional sources. Together, the proposed studies have the potential to reveal broadly relevant molecular mechanisms that are fundamental to c-di-GMP signaling and biofilm formation. Considering the central role of this process in infectious diseases, it is well accepted that understanding the underlying mechanisms may enable the development ...