Blatny J M, Brautaset T, Winther-Larsen H C, Haugan K, Valla S
UNIGEN Center for Molecular Biology, Norwegian University of Science and Technology, Trondheim, Norway.
Appl Environ Microbiol. 1997 Feb;63(2):370-9. doi: 10.1128/aem.63.2.370-379.1997.
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 xylR or xylS, 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.
本报告中描述的质粒载体源自广泛宿主范围的RK2复制子，可在多种革兰氏阴性细菌中维持。所有克隆和表达载体的完整核苷酸序列均已知。克隆载体的重要特征如下：大小范围为4.8至7.1 kb，具有独特的克隆位点，用于选择含质粒细胞的不同抗生素抗性标记，oriT介导的接合性质粒转移，质粒稳定功能，以及一种简单的调节质粒拷贝数的方法。表达载体是通过分别插入来自恶臭假单胞菌TOL质粒的可诱导的Pu或Pm启动子及其调节基因xylR或xylS构建而成。其中一种载体用于分析大肠杆菌中磷酸葡萄糖变位酶活性与直链淀粉积累之间的相关性。实验表明，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对直链淀粉合成的影响很小。相反，当诱导Pm转录时，直链淀粉积累大幅减少。CelB在野油菜黄单胞菌中也以非常高的诱导率表达。这些实验表明，木醋杆菌celB基因不能补充野油菜黄单胞菌双功能磷酸葡萄糖变位酶 - 磷酸甘露糖变位酶基因在黄原胶生物合成中的作用。我们认为，这里描述的载体可用于多种细菌的克隆实验、基因表达和生理学研究，大概也可用于环境中基因转移的分析。
