Cholera is a diarrheal disease caused by the Gram-negative bacterium Vibrio cholerae. To reach the surface of intestinal epithelial cells, proliferate, and cause disease, V. cholerae tightly regulates the production of virulence factors such as cholera toxin and the toxin co-regulated pilus (tcpA-F). The membrane-localized proteins TcpP and ToxR have been shown to play a role in this regulation pathway; however, the precise mechanism remains unclear. Recently, we quantified the interactions of TcpP and ToxR with the toxT promoter with live-cell single-molecule imaging and tracking. These experiments determined the cooperativity under steady-state conditions, but conventional sample preparation was incompatible with real-time perturbations. Therefore, we were unable to monitor dynamic changes during environment changes. Chitosan slide coatings are an excellent solution to this limitation as they enable media transfer during an experiment. I developed a method for bacterial cell immobilization and single-molecule imaging on chitosan-coated slides. This combination of slide coating and single-molecule fluorescence techniques enables us to observe the real-time response of the toxin expression pathway to environmental changes by monitoring quorum sensing in response to bile salts, stress from changes in pH, and dimerization of membrane proteins.

Achieving single-molecule tracking of subcellular regulation in bacteria during real-time environmental perturbations

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