Pristinamycin-inducible gene regulation in mycobacteria
Pristinamycin-inducible gene regulation in mycobacteria
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DOI：
10.1016/j.jbiotec.2009.02.001
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发表时间：
2009-03-25
影响因子：
4.1
通讯作者：
Ghisotti, Daniela
中科院分区：
文献类型：
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作者：
Forti, Francesca;Crosta, Andrea;Ghisotti, Daniela
在这项工作中，PIP诱导系统，已经在真核生物中使用，在分枝杆菌中进行了测试。该系统是基于天蓝色链霉菌Pip阻遏物、原始旋链霉菌ptr启动子和诱导物原始霉素1。通过在整合质粒中克隆lacZ报告基因上游的ptr启动子和在组成型分枝杆菌启动子控制下的pip基因，我们证明了ptr启动子活性在耻垢分枝杆菌中增加高达50倍，在结核分枝杆菌中增加高达400倍，这取决于原始霉素I浓度，在pptr-Pip控制下克隆了三个分枝杆菌基因，包括有义和反义方向：蛋白质和反义RNA都可以过表达，反义RNA导致靶蛋白量的部分减少。该系统用于获得两个M。fadD 32和pknB基因中的结核病条件突变体：突变菌株仅在诱导剂普那霉素1存在下生长。因此，它被证明是分枝杆菌中有效的诱导系统。(C)2009爱思唯尔有限公司版权所有。
In this work the Pip-inducible system, already used in eukaryotes, was tested in mycobacteria. This system is based on the Streptomyces coelicolor Pip repressor, the Streptomyces pristinaespiralis ptr promoter and the inducer pristinamycin 1. By cloning in an integrative plasmid the ptr promoter upstream of the lacZ reporter gene and the pip gene under the control of a constitutive mycobacterial promoter, we demonstrated that the ptr promoter activity increased up to 50-fold in Mycobacterium smegmatis and up to 400-fold in Mycobacterium tuberculosis, in dependence on pristinamycin I concentration, and that the promoter was fully repressed in the absence of the inducer.Three mycobacterial genes were cloned under pptr-Pip control, both in sense and antisense direction: both proteins and antisense RNAs could be over-expressed, the antisenses causing a partial reduction of the amount of the targeted proteins. This system was used to obtain two M. tuberculosis conditional mutants in the fadD32 and pknB genes: the mutant strains grew only in the presence of the inducer pristinamycin 1. Thus it showed to be an effective inducible system in mycobacteria. (C) 2009 Elsevier B.V. All rights reserved.