Fig. 4. LqqE1 targets multiple AHL synthases via physical interactions.
A Bacterial two-hybrid assay showing the direct binding between LqqE1 and PcoI but not PcoR. PcoI, the AHL synthase of P. fluorescens 2P24; PcoR, a neighbor of PcoI, is a LuxR-type receptor protein of AHL. Pull-down assay confirmed the direct binding of LqqE1-FLAG with PcoI-His (B) but not PcoR-His (C). Immunoprecipitation (IP) was carried out using anti-FLAG antibody and western blot was performed using anti-FLAG and anti-His antibodies. D Microscale thermophoresis (MST) showed MBP-LqqE1 bound to PcoI-His with moderately strong affinity (Kd, 330 nM). The lqqE1 expression in Pseudomonas aeruginosa PAO1 remarkably inhibited the RhlI-dependent (E), but not the LasI-dependent (F) AHL production. Both RhlI and LasI are AHL synthases of P. aeruginosa, and they are responsible for generating butyl-homoserine lactone (C4-HSL) and N-(3-oxododecanoyl)-L-homoserine lactone (3-oxo-C12-HSL), respectively. G Co-expression of the lqqE1 gene with the AHL synthase rhlI gene of P. aeruginosa PAO1 in E. coli significantly reduced AHL production. P. s. stands for P. aeruginosa PAO1. H Pull-down assay showing the LqqE1-FLAG bound to RhlI-His. The lqqE1 expression in P. aeruginosa PAO1 remarkably impaired the production of pyrazocyanin (I) and rhamnoolipids (J), two RhlI-controlled functions. K The lqqE1 expression in Ralstonia solanacearum EP1 inhibited the RasI-dependent AHL production. RasI is the major AHL synthase encoded by R. solanacearum EP1. R. s. stands for R. solanacearum EP1. L The lqqE1 expression in R. solanacearum EP1 impaired the RasI-controlled swimming motility. M Co-expression of the lqqE1 gene with the EP1 AHL synthase rasI gene in E. coli reduced AHL production. N Pull-down assay showing the LqqE1-FLAG bound to RasI-His. In all panels, average data from three experiments was presented, ±SD. ***p < 0.0001, **p < 0.01, *p < 0.05, “ns” stands for not statistically significant. Assessed by one-way ANOVA.