Bacterial cells communicate using quorum sensing, a process that involves the exchange of signal molecules known as autoinducers. While autoinducers were once thought to be species specific, the Bassler lab has characterized a “universal” autoinducer called AI-2. Interdomain communication was discovered when mammalian cells were found to produce a mimic of AI-2 capable of binding to AI-2 receptors in Vibrio harveyi, inducing bioluminescence. While the structure of this mimic has not been reported, recent data suggest that the mimic is xylosone. Despite the absence of a known enzyme for xylosone production in mammalian cells, previous literature characterizes pyranose oxidases as potential xylosone synthases, found throughout fungi and bacteria. In this thesis, it is shown that a fungal pyranose oxidase produces an AI-2 mimic, as measured by both high resolution mass spectrometry and a bioactivity assay in V. harveyi. This research lays the foundation for future work that could provide insights into AI-2 mediated chemical communication between bacteria and eukaryotes.

Quorum sensing (QS) is a process of bacterial cell-to-cell communication that allows bacteria to coordinate group behaviors and serves as a trigger for bacteriophage lysis. Bacteriophages monitor host bacterial QS signals, called autoinducers (AIs); in response to AI accumulation, phages produce proteins known as smORFs which sequester and inactivate lytic repressor proteins (cI), resulting in lysis at high host cell density. The exact mechanisms underlying this process in vivo are unclear, particularly in polylysogens. Previous work has shown that cI repressor proteins are specific to their partner smORFs and vice versa, despite the predicted similarities between cI proteins. This thesis aims to uncover the molecular basis of binding specificity displayed between cI proteins and their smORF partners. I first purified cI protein constructs of the full length and truncated cI proteins of two bacteriophage phage species. I then performed a co-elution assay using these purified protein constructs and determined that the N terminal domain of both species retained binding activity and specificity for its partner smORF protein. Furthermore, I developed a bioluminescence assay to test libraries of mutagenized cI proteins for altered smORF binding activity. The identification of specificity in this binding domain has implications for phage-phage and phage-bacteria interactions in the environment.