An Optimized Method for Detecting AI-2 Signal Molecule by a Bioassay with Vibrio harveyi BB170

Autoinducer-2 (AI-2) is a cell-to-cell signaling molecule that is commonly detected by an AI-2 bioassay with the Vibrio harveyi reporter strain BB170. However, the assay is affected by certain factors that can lead to inaccurate test results. Thus, the objective of this study was to establish the optimum method for detecting AI-2 by evaluating the effect of culture conditions for BB170, the preparation of the positive control and test samples, and the time point for AI-2 detection. The results showed that: (1) the ability of BB170 cultured in MB medium to specifically bioluminate in response to AI-2 was significantly higher than in LB medium; (2) the cell-free culture supernatant of V. harveyi BB152 served as a positive control, which was prepared from MB medium containing kanamycin; (3) an optimal time point of 4 to 6 h after inoculation of the bacterial samples was determined when using freshly cultured BB170 bacteria (OD600 = 2.0) to detect AI-2 signal molecules; (4) the optimum time point to detect AI-2 activity in the culture supernatant was the logarithmic growth phase. In summary, our findings will improve the screening of AI-2 molecules among microorganisms, thereby aiding the functional analysis of this cell-to-cell signaling molecule.