Structural Basis of Specificity in a Quorum Sensing-Induced Phage Lytic Pathway

Abstract

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.