A stringently controlled expression system for analysing lateral gene transfer between bacteria
A stringently controlled expression system for analysing lateral gene transfer between bacteria
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DOI：
10.1046/j.1365-2958.1996.6411358.x
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发表时间：
1996-07-01
影响因子：
3.6
通讯作者：
Diaz, E
中科院分区：
文献类型：
--
作者：
Jaenecke, S;deLorenzo, V;Diaz, E
微生物间遗传信息的横向传递是微生物群落对环境变化具有显著适应性的主要动力。到目前为止，很少有关于自然生态系统中基因转移的信息，我们在这里提出了一种遗传电路，用于检测和定量在缺乏对宿主特异性表型的选择的情况下从限定的供体微生物到受体生物体的水平基因转移，该系统由一个工程lacZ（编码β-半乳糖苷酶）报告基因，其表达受基于Pr启动子-来自λ噬菌体的操纵子和编码IS 10转座酶功能的tnp基因的5 ′非编码前导区，该报告盒在重组微生物中的表达被在转录水平上工作的两个染色体编码的反式作用阻遏物完全关闭（来自λ噬菌体的CI-EK 117蛋白），并且在翻译水平上当报告元件通过任何机制转移到不同的宿主时，它逃脱阻遏并表达。该系统用恶臭假单胞菌验证，并且检测到报告元件的接合转移频率低至10（-6）。基因电路的模块化设计和广泛的宿主范围，与允许实时原位检测的生物标志物相结合，将有助于以非破坏性的方式监测环境中的基因流动。
The lateral transfer of genetic information among microorganisms is a major force driving the outstanding adaptability of microbial communities to environmental changes. Until now little information has been obtained on gene transfer in natural ecosystems, We present here a genetic circuit for detecting and quantifying horizontal gene transfer from a defined donor microorganism to recipient organisms in the absence of selection for a recipient-specific phenotype, The system consists of an engineered lacZ (encoding beta-galactosidase) reporter gene whose expression is controlled by a synthetic regulatory element based on a fusion between the Pr promoter-operator from lambda bacteriophage and the 5' non-coding leader region of the tnp gene encoding the IS10 transposase function, Expression of this reporter cassette in the recombinant microorganism is completely shut down by two chromosomally encoded trans-acting repressors working at the level of transcription (the CI-EK117 protein from the lambda phage), and at the level of translation (the antisense RNA-OUT of the IS10 element), When the reporter element is transferred to a different host by any mechanism, it escapes repression and becomes expressed, The system was validated with Pseudo-monas putida, and conjugational transfer frequencies of the reporter element as low as 10(-6) were detected, The modular design and broad host range of the genetic circuit, in combination with biomarkers which permit real-time in situ detection, will facilitate the monitor-ing of gene flow in a non-disruptive manner within the environment.