Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria
Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria
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DOI：
10.1128/aem.02296-13
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发表时间：
2013-11-01
影响因子：
4.4
通讯作者：
Francez-Charlot, Anne
中科院分区：
文献类型：
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作者：
Kaczmarczyk, Andreas;Vorholt, Julia A.;Francez-Charlot, Anne
可调节启动子是一种广泛应用的关键遗传工具。在这里，我们为鞘氨醇单胞菌提供了这样一个系统，这是一组在系统发育上不同的细菌，它们在生物修复方面的潜力和在工业上的应用引起了人们的极大兴趣，但到目前为止还没有专门的诱导基因表达系统被描述。首先通过遗传筛选确定了一个强大的结构性合成启动子，然后将其与恶臭假单胞菌F1 Cym/CMT系统的抑制子和操纵子结合。由此产生的启动子，称为P-Q5，对诱导剂Cumate反应迅速，在所测试的不同鞘氨酸单胞菌中，最大诱导率为2-3个数量级。此外，它在其他甲型变形菌中也有作用，如模式生物新月弯杆菌、脱氮拟球藻和甲基杆菌。在非诱导状态下，P-Q5的表达低到足以进行基因缺失分析，就像神经鞘氨醇单胞菌的必需基因PhyP所证明的那样。菌株Fr1。已经构建了一组基于P-Q5的质粒，可以与亲和标签或荧光蛋白融合。
Tunable promoters represent a pivotal genetic tool for a wide range of applications. Here we present such a system for sphingomonads, a phylogenetically diverse group of bacteria that have gained much interest for their potential in bioremediation and their use in industry and for which no dedicated inducible gene expression system has been described so far. A strong, constitutive synthetic promoter was first identified through a genetic screen and subsequently combined with the repressor and the operator sites of the Pseudomonas putida F1 cym/cmt system. The resulting promoter, termed P-Q5, responds rapidly to the inducer cumate and shows a maximal induction ratio of 2 to 3 orders of magnitude in the different sphingomonads tested. Moreover, it was also functional in other Alphaproteobacteria, such as the model organisms Caulobacter crescentus, Paracoccus denitrificans, and Methylobacterium extorquens. In the noninduced state, expression from P-Q5 is low enough to allow gene depletion analysis, as demonstrated with the essential gene phyP of Sphingomonas sp. strain Fr1. A set of P-Q5-based plasmids has been constructed allowing fusions to affinity tags or fluorescent proteins.