Charlton T S, de Nys R, Netting A, Kumar N, Hentzer M, Givskov M, Kjelleberg S
School of Microbiology and Immunology, University of New South Wales, Australia.
Environ Microbiol. 2000 Oct;2(5):530-41. doi: 10.1046/j.1462-2920.2000.00136.x.
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).
报道了一种定量检测3-氧代酰基高丝氨酸内酯（3-oxo AHLs）的方法，3-oxo AHLs是革兰氏阴性菌中发现的一类主要的群体感应信号分子。该方法基于将3-oxo AHLs转化为其五氟苄基肟衍生物，然后进行气相色谱-质谱联用（电子捕获负离子模式）分析。该方法使用[13C16]-N-3-氧代十二烷酰高丝氨酸内酯（[13C16]-OdDHL）作为内标，并通过在样品上清液和细胞组分中添加三种浓度水平的3-oxo AHLs（即每份样品1、10和100 ng）来测试其有效性。结果表明该方法既灵敏（1 ng时信噪比>10:1）又准确。该检测方法应用于在流动小室中培养的表达绿色荧光蛋白（GFP）的铜绿假单胞菌（6294）菌株的生物膜和流出液。通过共聚焦扫描激光显微镜对三个重复的流动小室中的生物膜体积进行了测定。在生物膜中检测到OdDHL的浓度为632±381 μM，在流出液中为14±3 nM。生物膜中的浓度是迄今为止在野生型细菌系统中报道的AHL的最高水平。生物膜和流出液中其次最丰富的3-oxo AHL是N-3-氧代十四烷酰高丝氨酸内酯（OtDHL），分别为40±15 μM和1.5±0.7 nM。OtDHL在铜绿假单胞菌中未被报道，并且在lasR生物测定中具有与OdDHL相当的活性。还检测到另外两种浓度较低的3-oxo AHL：生物膜中（3±2 μM）和流出液中（1±0.1 nM）的N3-氧代癸酰高丝氨酸内酯（ODHL）；以及流出液中（0.1±0.1 nM）的N-3-氧代辛酰高丝氨酸内酯（OOHL）。
