Many species of bacteria regulate gene expression in response to changes in cell-density. This process, known as quorum sensing, depends on the production, secretion, accumulation, and detection of diffusible signaling molecules called autoinducers (AIs). By monitoring the levels of autoinducers present in their environment, bacteria are capable of determining whether they exist in relative isolation or as part of a large population. In this way, an entire population of bacteria can simultaneously alter gene expression and achieve an appropriate response adapted for particular population densities.

The experiments described in this thesis were undertaken in an effort to understand how quorum sensing operates in two related bacterial species that employ a diverse assortment of signaling molecules and modes of detection. The investigation of quorum sensing in the human pathogen Vibrio cholerae centers on the production of a species-specific signal, known as cholera autoinducer-1 (CAI-1), that controls virulence factor and toxin production. The only gene known to be required for CAI-1 production is cqsA. This work establishes that the product of cqsA is a pyridoxal-5’-phosphate (PLP)-dependent enzyme with aminotransferase activity. The CqsA enzyme ligates (S)-2-aminobutyric acid and decanoyl-coenzyme A to synthesize a precursor of CAI-1. This analysis of CqsA also reveals an apparent necessity for at least one additional, as yet unidentified enzyme to be involved in conversion of this precursor to the ultimate CAI-1 product.

This thesis also examines detection of the atypical autoinducer-2 (AI-2) by Vibrio harveyi. Whereas most autoinducers are species-specific signals, AI-2 is produced and detected by a large number of different bacterial species. V. harveyi uses the transmembrane receptor complex LuxPQ to detect AI-2 and to regulate phosphate flow through a downstream signaling cascade, ultimately controlling gene expression. The structural studies described here demonstrate that the periplasmic domain of the hybrid sensor kinase LuxQ does not change in response to binding of AI-2 to the LuxPQ complex. This result provides evidence for a model of transmembrane signaling that invokes quaternary rearrangement between LuxQ subunits in response to AI-2 binding rather than internal, tertiary conformational changes.

Taken together, this research into the intricacies of autoinducer synthesis in V. cholerae and autoinducer detection by V. harveyi contribute to both our understanding of bacterial quorum sensing—and to our appreciation of how complicated and awe-inspiring these seemingly simple organisms truly are.