Journal Article

Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography

Daly, S.L, Shaw, P.D. (University of Illinois, Urbana.), Farrand, S.K, Cha, C, Rinehart, K.L, Ping, G, Cronan, J.E. Jr

1997

Overview

Many Gram-negative bacteria regulate gene expression in response to their population size by sensing the level of acyl-homoserine lactone signal molecules which they produce and liberate to the environment. We have developed an assay for these signals that couples separation by thin-layer chromatography with detection using Agrobacterium tumefaciens harboring lacZ fused to a gene that is regulated by autoinduction. With the exception of N-butanoyl-L-homoserine lactone, the reporter detected acyl-homoserine lactones with 3-oxo-, 3-hydroxy-, and 3-unsubstituted 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 produce as many as five detectable signal molecules. Structures could be assigned tentatively on the basis of mobility and spot shape. The dominant species produced by Pseudomonas syringae pv. tabaci chromatographed with the properties of N-(3-oxohexanoyl)-L-homoserine lactone, a structure that was confirmed by mass spectrometry. An isolate of Pseudomonas fluorescens produced five detectable species, three of which had novel chromatographic properties. These were identified as the 3-hydroxy- forms of N-hexanoyl-, N-octanoyl-, and N-decanoyl-L-homoserine lactone. The assay can be used to screen cultures of bacteria for acyl-homoserine lactones, for quantifying the amounts of these molecules produced, and as an analytical and preparative aid in determining the structures of these signal molecules

• Publisher: National Academy of Sciences of the United States of America,National Acad Sciences,National Academy of Sciences,The National Academy of Sciences of the USA
• Subject: 4-Butyrolactone / 4-Butyrolactone - analogs & derivatives / 4-Butyrolactone - analysis / Acetates / ADN RECOMBINADO / ADN RECOMBINE / AGENT PATHOGENE / Agrobacterium radiobacter / AGROBACTERIUM TUMEFACIENS / Agrobacterium tumefaciens - genetics / Agrobacterium tumefaciens - metabolism / analogs & derivatives / analysis / assays / Bacteria / BACTERIA GRAM NEGATIVA / BACTERIE GRAM NEGATIF / BACTERIOSE / BACTERIOSIS / BETA GALACTOSIDASA / BETA GALACTOSIDASE / beta-Galactosidase - biosynthesis / Biochemistry / Biological Sciences / BIOSINTESIS / BIOSYNTHESE / biosynthesis / CHROMATOGRAPHIE EN COUCHE MINCE / Chromatography / Chromatography, Thin Layer / Chromatography, Thin Layer - methods / Cloning, Molecular / CROMATOGRAFIA DE CAPA FINA / Ensifer meliloti / EXPERIMENTACION / EXPERIMENTATION / GENE / Gene Expression Regulation, Bacterial / GENES / genetics / Gram-negative bacteria / Gram-Negative Bacteria - genetics / Gram-Negative Bacteria - metabolism / LACTONAS / LACTONE / Lactones / Mass spectroscopy / metabolism / methods / Molecules / ORGANISMOS PATOGENOS / plant pathogenic bacteria / Pseudomonas / Pseudomonas - genetics / Pseudomonas - metabolism / Pseudomonas fluorescens / Pseudomonas fluorescens - genetics / Pseudomonas fluorescens - metabolism / recombinant DNA / Recombinant Fusion Proteins / Recombinant Fusion Proteins - biosynthesis / reporter genes / RHIZOBIUM MELILOTI / Signal detection / Signal Transduction / Sinorhizobium meliloti / Solvents / Spectrometry, Mass, Secondary Ion / Thin layer chromatography