Construction 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. For detecting AHLs and indicating the host cells in situ, we constructed the plasmid pUCGMA2T(1-4) to make a dual fluorescent whole-cell biosensor based on the AhlIR AHL system of Pseudomonas syringae pv. syringae B728a. The plasmid contains three components: constitutively expressed P(npatII::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 x 10(-8)-1 x 10(-5) mol/L of 3OC6-HSL. Bright green fluorescence indicated the host cells. Furthermore, when the plasmid was transferred to wildtype 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.