Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi.
Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi.
复制标题
DOI：
10.1073/pnas.080068797
复制
发表时间：
2000-04
影响因子：
11.1
通讯作者：
M. Sartakova;E. Dobrikova;F. Cabello
中科院分区：
文献类型：
--
作者：
M. Sartakova;E. Dobrikova;F. Cabello
莱姆病的病因伯氏疏螺旋体的分子遗传学分析由于缺乏任何有效产生、鉴定和互补染色体和质粒无效基因突变体的手段而受到阻碍。疏螺旋体的G + C含量的相似性，革兰氏阳性菌表明，广泛的宿主范围的质粒活性革兰氏阳性菌也可能被疏螺旋体的DNA复制机制。一种这样的质粒pGK12能够在革兰氏阳性和革兰氏阴性细菌中繁殖，并携带红霉素和氯霉素抗性标记。pGK 12在B中染色体外增殖。burgdorferi B31，但仅赋予红霉素抗性。pGK 12用于在flaB启动子的控制下在B31中表达增强的绿色荧光蛋白。用从仅表达红霉素抗性的B31中提取的pGK 12 DNA转化大肠杆菌，产生红霉素和氯霉素抗性，并且从这些转化的大肠杆菌中分离质粒DNA。大肠杆菌的酶切图谱与原始pGK 12相似。我们的数据表明pGK 12的复制子可以为继续开发有效的B遗传系统提供基础。与红霉素抗性和报告基因EGFP一起。
Molecular genetic analysis of Borrelia burgdorferi, the cause of Lyme disease, has been hampered by the absence of any means of efficient generation, identification, and complementation of chromosomal and plasmid null gene mutants. The similarity of borrelial G + C content to that of Gram-positive organisms suggested that a wide-host-range plasmid active in Gram-positive bacteria might also be recognized by borrelial DNA replication machinery. One such plasmid, pGK12, is able to propagate in both Gram-positive and Gram-negative bacteria and carries erythromycin and chloramphenicol resistance markers. pGK12 propagated extrachromosomally in B. burgdorferi B31 after electroporation but conferred only erythromycin resistance. pGK12 was used to express enhanced green fluorescent protein in B31 under the control of the flaB promoter. Escherichia coli transformed with pGK12 DNA extracted from B31 expressing only erythromycin resistance developed both erythromycin and chloramphenicol resistance, and plasmid DNA isolated from these transformed E. coli had a restriction pattern similar to the original pGK12. Our data indicate that the replicons of pGK12 can provide the basis to continue developing efficient genetic systems for B. burgdorferi together with the erythromycin resistance and reporter egfp genes.