Mar 10, 2001

A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry: application to a model bacterial biofilm

Environmental Microbiology
T S CharltonStaffan Kjelleberg

Abstract

A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gram-negative bacteria. It is based on the conversion of 3-oxo AHLs to their pentafluorobenzyloxime derivatives followed by gas chromatography-mass spectrometry (electron capture-negative ion). The method used [13C16]-N-3-oxo-dodecanoyl homoserine lactone ([13C16]-OdDHL) as the internal standard, and its validity was tested by spiking the supernatant and cell fractions with three levels of 3-oxo AHLs, i.e. 1, 10 and 100 ng per sample. These showed the method to be both sensitive (S/N ratio >10:1 for 1 ng) and accurate. The assay was applied to the biofilm and effluent of a green fluorescent protein (GFP)-expressing strain of Pseudomonas aeruginosa (6294) culture grown in flow cells. Biofilm volume was determined for three replicate flow cells by confocal scanning laser microscopy. OdDHL was detected in the biofilm at 632 +/- 381 microM and the effluent at 14 +/- 3 nM. The biofilm concentration is the highest level so far reported for an AHL in a wild-type bacterial system. The next most abundant 3-oxo AHL in the biofilm and effluent was N-3-oxo-tetradecanoyl homoserine lactone (OtDHL) at 40...Continue Reading

Mentioned in this Paper

L-isomer of Homoserine
Pseudomonas aeruginosa (antigen)
Derivatives
Laser Microscopy
Anterior Horizontal Limb of Lateral Sulcus (Human Only)
Gas Chromatography-Mass Spectrometry
Lactones
Fluorescent stain
Gram-Negative Bacteria
Supernatant

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