Methods for detecting acylated homoserine lactones produced by Gram-negative bacteria and their application in studies of AHL-production kinetics

In the process of evaluating the role of acylated homoserine lactones (AHLs) in food-spoiling Gram-negative bacteria, we have combined a range of bacterial AHL monitor systems to determine the AHL-profile and the kinetics of AHL-production. AHL production from 148 strains of Enterobacteriaceae isolated from foods was tested using Escherichia coli pSB403 (LuxR), Agrobacterium tumefaciens A136 (TraR) and both induction and inhibition of Chromobacterium violaceum CV026 (CviR). All strains except one was found to produce AHL(s). In no case could a single monitor system identify more than 64% of the Enterobacteriaceae as AHL-producers, showing that the simultaneous use of monitor strains is required in the process of screening bacterial populations for AHL-production. AHLs from 20 selected strains were profiled by thin layer chromatography. Most strains produced more than one AHL with 3-N-oxo-hexanoyl homoserine lactone being the most prominent. It was found that the simultaneous use of monitor strains in the top-layer was necessary for the detection of (presumably) all the AHLs. An agar well-diffusion assay based on A. tumefaciens pDZLR4 was used for quantifying AHLs from bacterial supernatants and enabled an assessment of the kinetics of AHL-production of 3 strains (Serratia proteamaculans strain B5a, Erwinia carotovora ATCC 39048 and V. fischeri strain MJ-1). As expected, the production of AHL (OHHL) and luminescence in Vibrio fischeri strain MJ-1 increased faster than growth indicating up-regulation of the AHL regulated phenotype and auto-induction of AHL production. In contrast, production kinetics of AHL (OHHL) in the two Enterobacteriaceae indicated lack of auto-induction.