DETECTING AND CHARACTERIZING N-ACYL-HOMOSERINE LACTONE SIGNAL MOLECULES BY THIN-LAYER CHROMATOGRAPHY

Many Gram-negative bacteria regulate gene expression in response to th eir population size by sensing the level of acyl-homoserine lactone si gnal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by t hin-layer chromatography with detection using Agrobacterium tumefacien s harboring lacZ fused to a gene that is regulated by autoinduction, W ith the exception of N-butanoyl-L-homoserine lactone, the reporter det ected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubsti tuted side chains of all lengths tested. The intensity of the response was proportional to the amount of the signal molecule chromatographed . Each of the 3-oxo- and the 3-unsubstituted derivatives migrated with a unique mobility, Using the assay, we showed that some bacteria prod uce as many as five detectable signal molecules, Structures could be a ssigned tentatively on the basis of mobility and spot shape, The domin ant species produced by Pseudomonas syringae pv. tabaci chromatographe d with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a str ucture that was confirmed by mass spectrometry, An isolate of Pseudomo nas fluorescens produced five detectable species, three of which had n ovel chromatographic properties. These were identified as the 3-hydrox y- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lact one. The assay can be used to screen cultures of bacteria for acyl-hom oserine lactones, for quantifying the amounts of these molecules produ ced, and as an analytical and preparative aid in determining the struc tures of these signal molecules.