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

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 +/- 15 microM and 1.5 +/- 0.7 nM respectively. OtDHL is unreported for P. aeruginosa and has an activity equivalent to OdDHL in a lasR bioassay. Two other 3-oxo AHLs were detected at lower concentrations: N3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 microM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).

The role of quorum sensing signalling in EPS production and the assembly of a sludge community into aerobic granules

“…These observations are consistent with a previous report showing that AHLs preferentially partition to the biofilm, reaching concentrations 600-fold in excess of the signal concentration in the surrounding bulk liquid medium (Charlton et al, 2000). Thus, it is highly likely that QS induction would be active in the floccular and granular biomass tested here, and may be important in the development of microbial granules.…”

Nasal chemosensory cells use bitter taste signaling to detect irritants and bacterial signals

“…2D). Because bacterially produced biofilms generate levels of AHL in excess of 200 μM (14), this detection level is well within the range of concentrations predicted for an expanding bacterial population.…”

“…[11][12][13]. When population density is low, the concentration of AHLs is low; as the population density increases, the AHL concentration also increases, reaching levels over 200 μM (14). This triggers downstream signaling cascades in the bacteria through specific AHL-binding transcriptional regulatory proteins leading to the production of pathogenic biofilms and destructive virulence factors (15).…”

Production of acylated homoserine lactone by a novel marine strain of Proteus vulgaris and inhibition of its swarming by phytochemicals

“…The production of OHL was thus adjudged to be growth-associated. At 24 h, the Proteus vulgaris BNW culture produced 5.67 mg OHL l 21 (5.9 mM), which is in accordance with values reported in the literature (Charlton et al, 2000).…”