A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry:: application ito a model bacterial biofilm
A novel and sensitive method for the quantification of N-3-oxoacyl homoserine lactones using gas chromatography-mass spectrometry:: application ito a model bacterial biofilm
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DOI：
10.1046/j.1462-2920.2000.00136.x
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发表时间：
2000-10-01
影响因子：
5.1
通讯作者：
Kjelleberg, S
中科院分区：
文献类型：
--
作者：
Charlton, TS;de Nys, R;Kjelleberg, S
报道了一种用于定量3-氧代酰基高丝氨酸内酯（3-oxoacyl homoserine lactones，3-oxoAHLs）的方法，3-oxoAHLs是在革兰氏阴性菌中发现的一类主要的群体感应信号。它是基于3-氧代AHLs转化为其五氟苄肟衍生物，然后进行气相色谱-质谱分析（电子捕获-负离子）。该方法使用[C-13（16）]-N-3-氧代-十二烷酰基高丝氨酸内酯（[C-13（16）]-OdDML）作为内标，并通过向上清液和细胞组分中加入三个水平的3-氧代AHL（即每个样品1、10和100 ng）来测试其有效性，这表明该方法既灵敏（1 ng的S/N比> 10：1）又准确。将该测定应用于在流动池中生长的铜绿假单胞菌（6294）培养物的绿色荧光蛋白（GFP）表达菌株的生物膜和流出物。通过共聚焦扫描激光显微镜测定三个重复流动池的生物膜体积。在生物膜中检测到632 +/-381 μ M的OdDHL，在流出物中检测到14 +/-3 nM的OdDHL。生物膜浓度是迄今为止报道的野生型细菌系统中阿勒的最高水平。生物膜和流出物中第二丰富的3-氧代阿勒是N-3-氧代-十四酰基高丝氨酸内酯（OtDHL），分别为40 +/-15 μ M和1.5 +/-0.7 nM。未报告OtDHL对铜绿假单胞菌的作用，其活性与lasR生物测定中的OdDHL相当。在较低浓度下检测到另外两种3-氧代AHL：生物膜（3 +/-2 μ M）和流出物（1 +/-0.1 nM）中的N-3-氧代-癸酰基高丝氨酸内酯（ODHL）;和流出物（0.1 +/-0.1 nM）中的N-3-氧代-辛酰基高丝氨酸内酯（OOHL）。
A method is reported for the quantification of 3-oxoacyl homoserine lactones (3-oxo AHLs), a major class of quorum-sensing signals found in Gramnegative 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 [C-13(16)]-N-3-oxo-dodecanoyl homoserine lactone ([C-13(16)]-OdDML) 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 aeroginosa (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 muM 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 muM 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: N-3-oxo-decanoyl homoserine lactone (ODHL) in the biofilm (3 +/- 2 muM) and effluent (1 +/- 0.1 nM); and N-3-oxo-octanoyl homoserine lactone (OOHL) in the effluent (0.1 +/- 0.1 nM).