Leenhouts K J, Kok J, Venema G
Institute of Genetics, University of Groningen, Haren, The Netherlands.
Appl Environ Microbiol. 1989 Feb;55(2):394-400. doi: 10.1128/aem.55.2.394-400.1989.
Integrable vectors were constructed based on the plasmid pHV60, which is essentially a pBR322 replicon carrying a chloramphenicol resistance marker, by inserting 1.3-kilobase chromosomal fragments of Lactococcus lactis subsp. lactis MG1363 into this plasmid. Three constructs as well as pHV60 were electroporated to strain MG1363. Transformants were obtained with all constructs, and also with pHV60 (albeit with low frequency). By using Southern hybridizations, it appeared that pHV60 showed homology with the chromosome of MG1363, and that it most probably uses this homology to integrate in a Campbell-like manner. The presence of chromosomal sequences in pHV60 stimulated insertion elsewhere in the chromosome by a factor of 5 to 100. In all cases the integrated plasmids were amplified, at a selective pressure of 5 micrograms of chloramphenicol per ml, to a level of approximately 15 copies per chromosome. Although the amplification was gradually lost under nonselective conditions, one copy remained stably integrated in the chromosome. The results show that a Campbell-like integration strategy can be used to improve the accessibility of the lactococcal chromosome for genetic analysis and is potentially useful in stabilizing unstable genes in lactococci.
基于质粒pHV60构建了可整合载体，该质粒本质上是携带氯霉素抗性标记的pBR322复制子，通过将乳酸乳球菌乳亚种MG1363的1.3千碱基染色体片段插入该质粒中。将三个构建体以及pHV60电穿孔导入菌株MG1363。所有构建体以及pHV60（尽管频率较低）均获得了转化子。通过Southern杂交发现，pHV60与MG1363的染色体显示出同源性，并且它很可能利用这种同源性以坎贝尔式的方式进行整合。pHV60中染色体序列的存在使染色体其他位置的插入增加了5到100倍。在所有情况下，整合的质粒在每毫升5微克氯霉素的选择压力下扩增至每条染色体约15个拷贝的水平。尽管在非选择条件下扩增逐渐丧失，但仍有一个拷贝稳定地整合在染色体中。结果表明，坎贝尔式的整合策略可用于提高乳酸乳球菌染色体用于遗传分析的可及性，并且在稳定乳酸乳球菌中不稳定基因方面可能有用。
