Abstract: We study the molecular mechanisms and signaling, from environmental input to physiological output, that control bacterial biofilm formation, a key bacterial lifestyle linked to host-microbe interactions and infectious disease. In particular, the capability of bacteria to sense and respond to various microenvironments, particularly during the transition from a free-swimming to a sessile biofilm lifestyle, contributes to the establishment of chronic infections. The underlying mechanisms are equally important for non-pathogenic bacteria that can live in commensal relationship with their host(s). Understanding the architecture and regulation of signaling systems that control bacterial cell adhesion and biofilm formation is critical in the development of novel therapies and preventative interventions, while providing fundamental insight into bacterial signaling processes. Here, we propose mechanistic studies on a broadly conserved cell adhesion system that is crucial for biofilm formation in a variety of pathogenic and commensal organisms. We will focus on fundamental unanswered questions concerning how cyclic-di-GMP networks control localization of these adhesins as well as how two such adhesins contribute to formation of biofilms. We will also enhance the impact of our studies by investigating a newly identified, analogous signaling system in an important commensal sulfate- reducing bacterium. The central hypothesis of this proposal is that cyclic-di-GMP signaling via a network of ligand-responsive DGCs regulates biofilm formation across a number of microbes of importance in pathogenic, host-associated, and environmental contexts. We propose the following Specific Aims to test this hypothesis: AIM 1. Test the hypothesis that small-molecule ligands are critical for regulating the localized cdG network via receptor complexes in P. fluorescens. AIM 2. Test the hypothesis that discrete domain structures of LapA and MapA in Pfl contribute to their differential impacts on biofilm formation. AIM 3. Test the hypothesis that the sulfate-reducing bacterium LapD/LapG-like system controls localization of this intestinal bacterium’s LapA homolog.

Project Narrative Signaling through the bacterial second messenger c-di-GMP is subject to exquisite control, especially in bacterial pathogens, which often simultaneously express a multitude of signaling components for setting cellular and local c-di-GMP levels and triggering distinct physiological responses, for example, controlling the expression of multiple adhesins critical for establishing a biofilm. The proposed studies will elucidate both intra- and inter-molecular modes of regulation of bacterial c-di-GMP signaling, explore the role for adhesins and their sub-domains in biofilm formation, and extend our work to a new and unusual Lap-like system in a human-associated microbe.