Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography
Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography
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DOI：
10.1073/pnas.94.12.6036
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发表时间：
1997-06-10
影响因子：
11.1
通讯作者：
Farrand, SK
中科院分区：
文献类型：
--
作者：
Shaw, PD;Ping, G;Farrand, SK
许多革兰氏阴性细菌通过感知它们产生并释放到环境中的酰基高丝氨酸内酯信号分子的水平来调节基因表达，以响应它们的种群大小。我们已经开发了一种用于这些信号的测定法，该测定法通过薄层色谱法与使用根癌农杆菌的检测进行分离，所述根癌农杆菌具有与受自诱导调节的基因融合的lacZ。除了N-丁酰基-L-高丝氨酸内酯之外，报告者检测到具有3-氧代-、3-羟基-和3-未取代侧链的所有测试长度的酰基-高丝氨酸内酯。响应的强度与色谱分离的信号分子的量成比例。3-氧代和3-未取代的衍生物都有独特的迁移率。利用这种方法，我们发现一些细菌产生多达5个可检测的信号分子，根据迁移率和斑点形状可以初步确定结构。利用N-（3-氧代己酰基）-L-高丝氨酸内酯的性质对烟粉虱进行色谱分析，该结构通过质谱法确认。荧光假单胞菌的分离物产生五种可检测的种类，其中三种具有新的色谱性质。这些被鉴定为N-己酰基-、N-辛酰基-和N-癸酰基-L-高丝氨酸内酯的3-羟基-形式。该测定可用于筛选酰基高丝氨酸内酯的细菌培养物，用于定量产生的这些分子的量，并作为确定这些信号分子结构的分析和制备辅助。
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.