Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing
Broad-host-range expression vectors with tightly regulated promoters and their use to examine the influence of TraR and TraM expression on Ti plasmid quorum sensing
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DOI：
10.1128/aem.01098-08
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发表时间：
2008-08-01
影响因子：
4.4
通讯作者：
Farrand, Stephen K.
中科院分区：
文献类型：
--
作者：
Khan, Sharik R.;Gaines, Jennifer;Farrand, Stephen K.
由于目前使用的启动子的基础表达水平相对较高，需要强阻遏和精确控制克隆基因的实验可能难以进行。我们报告了一系列载体的构建，这些载体包含重新设计的lacI（q）-lac启动子-操纵基因复合物，其中克隆的基因在没有诱导剂的情况下被强烈抑制。载体，所有的基础上广泛的主机范围质粒pBBR 1，是可移动的，并在中等拷贝数的α和γ变形菌的代表稳定复制。每个载体都含有一个通用的多克隆位点，其中包括一个NdeI位点，允许克隆基因与lacZ α的起始密码子融合。在每种测试的细菌中，与启动子融合的uidA报告基因在不存在诱导的情况下不以可检测的水平表达，但可诱导10至100倍，这取决于细菌。诱导的程度可通过改变诱导剂的浓度来控制。当载体在根癌农杆菌中进行测试时，traR基因的克隆拷贝，其产物在每个细胞中仅需要几个拷贝，在非诱导条件下不赋予活性。我们使用这种非常紧密和可调控的控制属性来评估激活Ti质粒接合转移系统所需的TraR的相对量。我们确定了产生野生型转移频率的诱导水平，以及诱导相应较低转移频率的水平。我们还使用该系统来显示抗激活剂TraM设置tra基因激活所需的细胞内TraR水平。
Experiments requiring strong repression and precise control of cloned genes can be difficult to conduct because of the relatively high basal level of expression of currently employed promoters. We report the construction of a family of vectors that contain a reengineered lacI(q)-lac promoter-operator complex in which cloned genes are strongly repressed in the absence of inducer. The vectors, all based on the broad-host-range plasmid pBBR1, are mobilizable and stably replicate at moderate copy number in representatives of the alpha- and gammaproteobacteria. Each vector contains a versatile multiple cloning site that includes an NdeI site allowing fusion of the cloned gene to the initiation codon of lacZ alpha. In each tested bacterium, a uidA reporter fused to the promoter was not expressed at a detectable level in the absence of induction but was inducible by 10- to 100-fold, depending on the bacterium. The degree of induction was controllable by varying the concentration of inducer. When the vector was tested in Agrobacterium tumefaciens, a cloned copy of the traR gene, the product of which is needed at only a few copies per cell, did not confer activity under noninducing conditions. We used this attribute of very tight and variably regulatable control to assess the relative amounts of TraR required to activate the Ti plasmid conjugative transfer system. We identified levels of induction that gave wild-type transfer frequencies, as well as levels that induced correspondingly lower frequencies of transfer. We also used this system to show that the antiactivator TraM sets the level of intracellular TraR required for tra gene activation.