Defining Agonism and Antagonism in Vibrio harveyi LuxN* Mutants

Abstract

Quorum sensing (QS) is a process of bacterial cell-to-cell communication that functions by the production and detection of extracellular autoinducer signaling molecules. In the marine bacterium Vibrio harveyi, LuxN is one of three QS receptors found on the inner cell membrane. The binding of LuxN to its autoinducer HAI-1, synthesized by LuxM, causes a switch from kinase to phosphatase activity and subsequently activates QS gene expression. In this study, we made a series of LuxN point mutants, which are collectively referred to as LuxN* mutants. We examined six luxN* mutations (Leu166Ala, Leu166Val, His210Asn, Leu166Ala/His210Asn, Leu166Val/His210Asn, and Lys191Ala) expressed from V. harveyi plasmids and the influence they had on the free energy bias of LuxN* as well as the changes they made to LuxN* ligand specificity. We engineered these same mutations into the genomes of two V. harveyi background strains utilizing a novel combination of protocols to examine the biological relevance of endogenous LuxN* mutations on QS activity. Protein levels of chromosomally integrated LuxN were measured, and we also analyzed the binding of various agonists to the mutant LuxN* receptors. We discovered that our protocol was effective in transferring luxN* mutations from cosmids into the chromosome of V. harveyi. This led us to determine, through dose response experiments, that V. harveyi strains carrying mutant LuxN* altered the ligand specificity and/or the free energy state of the receptor and also affected an interplay between QS receptors LuxN and LuxPQ in activating downstream QS genes.