Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay
Analysing traces of autoinducer-2 requires standardization of the Vibrio harveyi bioassay
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DOI：
10.1007/s00216-006-0824-4
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发表时间：
2007-01-01
影响因子：
4.3
通讯作者：
Wagner-Doebler, Irene
中科院分区：
文献类型：
--
作者：
Vilchez, Ramiro;Lemme, Andre;Wagner-Doebler, Irene
自诱导物-2（呋喃糖基硼酸二酯）是一种生物活性化合物，其作为通用细菌信号分子的作用目前正在深入研究中。由于其不稳定性和在生物样品中发现的低浓度，其检测目前依赖于生物测定法，该生物测定法测量具有和不具有外部添加的AI-2的哈氏弧菌BB 170传感器菌株的发光时间的差异。在这里，我们系统地研究了哪些参数影响在生物测定中获得的发光的倍数诱导值，并开发了一个修改后的协议。实验结果表明，微量元素对哈维氏弧菌BB 170的生长和发光有很大的影响。特别是，在一定浓度范围内的Fe 3+添加到预接种物培养物的生长培养基中，提高了重现性并降低了生物测定的方差。相反，微量元素和维生素直接引入生物测定引起抑制作用。传感器应变的初始密度和发光是非常重要的，并且定义了这些参数所需的值。硼酸盐通过给出假阳性结果而干扰AI-2的检测。在生物测定中，哈维氏弧菌BB 170对化学合成的AI-2的响应是非线性的，除了在非常小的浓度范围内;它在三个数量级上是最大的，并且在35 μ M以上显示出抑制作用。基于修改的方案，我们能够在不存在抑制剂的情况下检测AI-2，阳性对照（化学合成的AI-2）的最大倍数诱导值> 120，标准偏差类似于30%，以可靠和可再现的方式。
Autoinducer-2 (furanosyl borate diester) is a biologically active compound whose role as a universal bacterial signalling molecule is currently under intense investigation. Because of its instability and the low concentrations of it found in biological samples, its detection relies at present on a bioassay that measures the difference in the timing of the luminescence of the Vibrio harveyi BB170 sensor strain with and without externally added AI-2. Here we systematically investigated which parameters affected the fold induction values of luminescence obtained in the bioassay and developed a modified protocol. Our experiments showed that growth and luminescence of V. harveyi BB170 are strongly influenced by trace elements. In particular, addition of Fe3+ within a certain concentration range to the growth medium of the preinoculum culture improved the reproducibility and reduced the variance of the bioassay. In contrast, trace elements and vitamins introduced directly into the bioassay caused inhibitory effects. The initial density and luminescence of the sensor strain are very important and the values required for these parameters were defined. Borate interferes with the detection of AI-2 by giving false positive results. The response of V. harveyi BB170 to chemically synthesized AI-2 in the bioassay is nonlinear except over a very small concentration range; it is maximum over three orders of magnitude and shows inhibition above 35 mu M. Based on the modified protocol, we were able to detect AI-2 in the absence of inhibitors with maximum fold induction values for the positive control (chemically synthesized AI-2) of > 120 with a standard deviation of similar to 30% in a reliable and reproducible way.