A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes
A plasmid-based genomic screening system for transcriptional regulators of non-adjacent xenobiotic catabolism genes
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基于质粒的基因组筛选系统，用于非相邻外源分解代谢基因的转录调节因子
DOI：
10.1007/s00253-019-10268-5
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发表时间：
2019-12
影响因子：
5
通讯作者：
Huang Xing
中科院分区：
文献类型：
--
作者：
Cheng Minggen;Xing Ziyu;Lu Luyao;Chen Feng;He Jian;Huang Xing
细菌在环境异生物质的催化过程中起着重要作用。对转录调控的研究极大地增强了我们对这些过程相关分子机制的理解。然而，基因编码的转录因子（TF）的异生物质分解酶通常不位于附近的分解代谢基因，它们调节，因此，这些TF的功能鉴定是困难的。从宏基因组筛选方法底物诱导的基因表达（SIGEX）显着修改，在这里，我们提出了一个合成的pSRGFP-18质粒为基础的工具作为TF报告系统。简而言之，设计了两个多克隆位点（MCS）;一个在anegfpreporter基因之前构建用于启动子插入，另一个MCS用于基因组DNA的鸟枪克隆。基于TF和其相关分解代谢基因的启动子之间的调控关系，这种方法使我们能够使用基因组鸟枪法筛选TF基因。该系统在测试已知操纵子时表现良好。根据对大肠杆菌和枯草芽孢杆菌中已知的分解代谢操纵子的统计分析，该系统的目标启动子的建议区域为-250到+150。此外，为了扩大该质粒的适用性，构建了一系列基于pSRGFP-18和pBBR 1的pSRGFP-X质粒，这些质粒具有广泛的宿主范围，并含有不同的抗生素标记。这项研究概述了一个强大的工具，使功能鉴定的TF细菌异生素催化剂。
Bacteria play an important role in the catabolism of environmental xenobiotics. The study of transcriptional regulation has greatly enhanced our understanding of the molecular mechanisms associated with these processes. However, genes encoding transcription factors (TFs) for xenobiotic catabolism are usually not located in the immediate vicinity of the catabolic genes that they regulate; therefore, functional identification of these TFs is difficult. Significantly modified from a metagenome screening method substrate-induced gene expression (SIGEX), here we propose a synthetic pSRGFP-18 plasmid-based tool as a TF reporter system. In short, two multiple cloning sites (MCS) were designed; one in front of anegfpreporter gene was constructed for promoter insertion, and the other MCS was used for shotgun cloning of genomic DNA. Based on the regulatory relationship between TFs and the promoter of their associated catabolic genes, this approach allowed us to screen for TF genes using a genome shotgun approach. This system performed well when testing the known operons. Following statistical analysis of known catabolic operons inEscherichia coliandBacillus subtilis, the suggested region of the target promoter for this system was from − 250 to + 150. Furthermore, to broaden the applicability of this plasmid, a series of pSRGFP-18 and pBBR1-based pSRGFP-X plasmids were constructed, which had broad host ranges and contained different antibiotic markers. This study outlines a powerful tool to enable functional identification of TFs for bacterial xenobiotic catabolism.
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