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Background Bacteria coordinate behavior with hormone-like chemicals called autoinducers. As a population grows, autoinducer concentration rises in step; past a threshold the cells sense the crowd and change behavior via quorum sensing. It underlies biofilms, bioreactor performance, and anti-virulence drug targets. Why Vibrio campbellii? Aliivibrio fischeri is a widely used model organism, which accommodates diffusion mediated signaling; however, signaling bypasses receptor mediated signaling. V. campbellii has the light (lux) operon controlled by membrane histidine-kinase receptors converging on one phosphorylation cascade, an ideal model for teaching signal transduction. Research Question: Can we turn an invisible protein cell signaling pathway into something students can see? Can we give students the agency to perturb this pathway, see the visible effect, and extrapolate why? Most importantly, can we do it safely? Problems: V. campbellii is not a human pathogen, but it is a marine pathogen. Vibriosis is a concern in aquaculture systems with high mortality rates (possible loss of an entire population). There are three major histidine kinase receptors complicating the ability to effectively interrogate the phosphorylation cascade. In line with our high standards for environmental stewardship, we created attenuated, highly bio contained the Vibrio strains. Design choices: - Focus on one stable, well-behaved receptor, LuxN, and its autoinducer HAI-1. This deleted the noisier LuxPQ and less influential CqsS receptors, so strains respond cleanly to a single ligand. - Attenuate and biocontain through auxotrophy + weakened DNA repair: ΔglmS, ΔthyA, ΔbioABFCD, ΔrecA. - Prophage element removal to remove risk of intact phage activation. - Kit-activity strains, ΔluxM, luxN-H471A, ΔluxB. With this and an upcoming related product, students can visualize and differentiate the signaling phases of reception, transduction, and response. Students understand the Lux pathway using a model. They culture bacteria, rescue luminescence, test inhibitors, and even design their own experiments.
