嗜盐古菌沃氏嗜盐碱杆菌严格调控启动子的特性及其在必需基因cct1分析中的应用

Characterization of a tightly controlled promoter of the halophilic archaeon Haloferax volcanii and its use in the analysis of the essential cct1 gene.

作者信息

Large Andrew, Stamme Claudia, Lange Christian, Duan Zhenhong, Allers Thorsten, Soppa Jörg, Lund Peter A

机构信息

School of Biosciences, The University of Birmingham, Edgbaston B15 2TT, UK.

出版信息

Mol Microbiol. 2007 Dec;66(5):1092-106. doi: 10.1111/j.1365-2958.2007.05980.x. Epub 2007 Oct 31.

Abstract

A system where archaeal gene expression could be controlled by simple manipulation of growth conditions would enable the construction of conditional lethal mutants in essential genes, and permit the controlled overproduction of proteins in their native host. As tools for the genetic manipulation of Haloferax volcanii are well developed, we set out to identify promoters with a wide dynamic range of expression in this organism. Tryptophan is the most costly amino acid for the cell to make, so we reasoned that tryptophan-regulated promoters might be good candidates. Microarray analysis of H. volcanii gene expression in the presence and absence of tryptophan identified a tryptophanase gene (tna) that showed strong induction in the presence of tryptophan. qRT-PCR revealed a very fast response and an up to 100-fold induction after tryptophan addition. This result has been confirmed using three independent reporter genes (cct1, pyrE2 and bgaH). Vectors containing this promoter will be very useful for investigating gene function in H. volcanii and potentially in other halophilic archaea. To demonstrate this, we used the promoter to follow the consequences of depletion of the essential chaperonin protein CCT1, and to determine the ability of heterologous CCT proteins to function in H. volcanii.

摘要

一个可以通过简单地控制生长条件来调控古菌基因表达的系统,将能够构建必需基因的条件致死突变体,并能在其天然宿主中可控地过量表达蛋白质。由于用于沃氏嗜盐菌(Haloferax volcanii)基因操作的工具已经很完善,我们着手在这种生物中鉴定具有广泛动态表达范围的启动子。色氨酸是细胞合成成本最高的氨基酸,因此我们推断色氨酸调控的启动子可能是很好的候选对象。对存在和不存在色氨酸时沃氏嗜盐菌基因表达的微阵列分析,鉴定出一个色氨酸酶基因(tna),该基因在色氨酸存在时表现出强烈的诱导作用。定量逆转录聚合酶链反应(qRT-PCR)显示出非常快速的反应,并且在添加色氨酸后诱导倍数高达100倍。使用三个独立的报告基因(cct1、pyrE2和bgaH)证实了这一结果。含有该启动子的载体对于研究沃氏嗜盐菌以及潜在地其他嗜盐古菌中的基因功能将非常有用。为了证明这一点,我们使用该启动子来追踪必需伴侣蛋白CCT1缺失的后果,并确定异源CCT蛋白在沃氏嗜盐菌中发挥功能的能力。

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