(584q) Development of a Dual Fluorescence Whole-Cell Biosensor to Detect N-Acyl Homoserine Lactones

Detection of N-acyl homoserine lactones (AHLs) is useful for understanding quorum sensing (QS) behaviors, including biofilm formation, virulence and metabolism. In this study, to not only detect AHLs but also to indicate the host cells in situ, the plasmid pUCGMA2T_1-4 was constructed to make a dual fluorescent whole-cell biosensor based on the AhlI/R AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components: constitutively expressed P_nptII::gfp for indicating host cells, P_ahlI::mcherry that produces red fluorescence in response to AHL, and the ahlR gene that encodes an AHL regulatory protein. Meanwhile, two copies of T_1-4 (four tandem copies of a transcriptional terminator) were added into the plasmid to reduce background. The results showed that when the plasmid was placed into Escherichia coli, the dual fluorescence whole-cell biosensor was able to respond with red fluorescence within 6 hr to 5×10⁻⁸−10⁻⁵ mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wild-type Pseudomonas PhTA125 (an AHL-producing bacterium), it also showed both green and red fluorescence. This result demonstrates that this plasmid can be used to construct whole-cell indicators that can indicate the AHL response and spatial behaviors of microbes in a microenvironmental niche.