Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon
Construction and use of a versatile set of broad-host-range cloning and expression vectors based on the RK2 replicon
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DOI：
10.1128/aem.63.2.370-379.1997
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发表时间：
1997-02-01
影响因子：
4.4
通讯作者：
Valla, S
中科院分区：
文献类型：
--
作者：
Blatny, JM;Brautaset, T;Valla, S
本报告中描述的质粒载体来源于广泛宿主范围的RK 2复制子，并可在许多革兰氏阴性细菌物种中维持。所有克隆和表达载体的完整核苷酸序列是已知的。克隆载体的重要特征如下：4.8至7.1 kb的大小范围、独特的克隆位点、用于选择含质粒细胞的不同抗生素抗性标记、oriT介导的接合质粒转移、质粒稳定化功能以及用于修饰质粒拷贝数的简单方法的手段。分别从恶臭假单胞菌的TOL质粒中插入诱导型Pu或Pm启动子及其调控基因xy/R或xy/S，构建表达载体。这些载体之一被用于在大肠杆菌中的磷酸葡萄糖变位酶活性和直链淀粉积累之间的相关性分析。实验表明，直链淀粉合成仅轻微地受木醋杆菌磷酸葡萄糖变位酶基因（celB）的Pm启动子的基础表达水平的影响。与此相反，直链淀粉积累强烈减少时，从Pm转录诱导。CelB在野油菜黄单胞菌（Xanthomonascampestris）中也有很高的诱导率。实验结果表明，A. xylinumcelB基因不能补充双功能X的作用。油菜磷酸葡萄糖变位酶-磷酸甘露糖变位酶基因在黄原胶生物合成中的作用。我们相信，这里描述的载体是有用的克隆实验，基因表达，和生理学研究与广泛的细菌，大概也为分析基因转移的环境。
The plasmid vectors described in this report are derived from the broad-host-range RK2 replicon and can be maintained in many gram-negative bacterial species. The complete nucleotide sequences of all of the cloning and expression vectors are known. Important characteristics of the cloning vectors are as follows: a size range of 4.8 to 7.1 kb, unique cloning sites, different antibiotic resistance markers for selection of plasmid-containing cells, oriT-mediated conjugative plasmid transfer, plasmid stabilization functions, and a means for a simple method for modification of plasmid copy number. Expression vectors were constructed by insertion of the inducible Pu or Pm promoter together with its regulatory gene xy/R or xy/S, respectively, from the TOL plasmid of Pseudomonas putida. One of these vectors was used in an analysis of the correlation between phosphoglucomutase activity and amylose accumulation in Escherichia coli. The experiments showed that amylose synthesis was only marginally affected by the level of basal expression from the Pm promoter of the Acetobacter xylinum phosphoglucomutase gene (celB). In contrast, amylose accumulation was strongly reduced when transcription from Pm was induced. CelB was also expressed with a very high induction ratio in Xanthomonas campestris. These experiments showed that the A. xylinum celB gene could not complement the role of the bifunctional X. campestris phosphoglucomutase-phosphomannomutase gene in xanthan biosynthesis. We believe that the vectors described here are useful for cloning experiments, gene expression, and physiological studies with a wide range of bacteria and presumably also for analysis of gene transfer in the environment.