Institute of Molecular Genetics and Genetic Engineering, Belgrade, Serbia.
J Appl Microbiol. 2009 Jan;106(1):78-88. doi: 10.1111/j.1365-2672.2008.03977.x. Epub 2008 Nov 21.
Strain Lactococcus lactis subsp. lactis bv. diacetylactis S50 harbours five theta-replicating plasmids (pS6, pS7a, pS7b, pS80 and pS140). The aim of this study was to characterize domains involved in the replication and conjugative mobilization of the small plasmids pS7a and pS7b, which are structurally very similar.
Complete nucleotide sequences of pS7a and pS7b were determined by cloning DNA fragments of different sizes into Escherichia coli vectors. Linearized plasmids and four EcoRI fragments of the pS7a and pS7b were cloned into an origin probe vector. Constructed plasmids (pSEV10, pSK10, pISE1a and pISE1b) were able to replicate in the strain L. lactis subsp. cremoris MG1363. In addition, experiments showed that plasmids pS7a and pS7b contained oriT sequences and their conjugative transfer directly depended on the presence of pS80 in donor cells.
Plasmids pS7a and pS7b contained typical lactococcal theta replication origin and repB gene that enable them to replicate in the strain L. lactis subsp. cremoris MG1363. Plasmid pS80 plays a key role in the conjugative transfer of small plasmids.
Plasmids pS7a and pS7b-based derivatives could be valuable tools for genetic manipulation, studying processes of plasmid maintenance and horizontal gene transfer in lactococci.
乳球菌亚种乳糖发酵双乙酰变种 S50 携带五个θ复制质粒（pS6、pS7a、pS7b、pS80 和 pS140）。本研究旨在鉴定与结构非常相似的小质粒 pS7a 和 pS7b 的复制和可接合移动相关的结构域。
通过将不同大小的 DNA 片段克隆到大肠杆菌载体中，确定了 pS7a 和 pS7b 的完整核苷酸序列。线性化质粒和 pS7a 和 pS7b 的四个 EcoRI 片段被克隆到一个原点探针载体中。构建的质粒（pSEV10、pSK10、pISE1a 和 pISE1b）能够在乳球菌亚种乳脂球菌 MG1363 中复制。此外，实验表明质粒 pS7a 和 pS7b 含有 oriT 序列，其可接合转移直接依赖于供体细胞中 pS80 的存在。
质粒 pS7a 和 pS7b 包含典型的乳球菌θ复制原点和 repB 基因，使它们能够在乳球菌亚种乳脂球菌 MG1363 中复制。质粒 pS80 在小质粒的可接合转移中起关键作用。
基于质粒 pS7a 和 pS7b 的衍生物可以成为遗传操作的有价值的工具，用于研究质粒在乳球菌中的维持和水平基因转移过程。
