HIGH-EFFICIENCY GENE INACTIVATION AND REPLACEMENT SYSTEM FOR GRAM-POSITIVE BACTERIA
HIGH-EFFICIENCY GENE INACTIVATION AND REPLACEMENT SYSTEM FOR GRAM-POSITIVE BACTERIA
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DOI：
10.1128/jb.175.11.3628-3635.1993
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发表时间：
1993-06-01
影响因子：
3.2
通讯作者：
MAGUIN, E
中科院分区：
文献类型：
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作者：
BISWAS, I;GRUSS, A;MAGUIN, E
以乳酸乳球菌（Lactococcus lactis）为模型系统，建立了革兰氏阳性菌单、双交换同源整合系统。该系统是基于一个温敏的宽宿主范围滚环质粒，pG+ host 5，其中含有一个pBR 322复制子，用于在37 ℃下在大肠杆菌中繁殖。L的嵌套集合。使用克隆到pG+宿主5上的乳酸菌染色体片段来显示单交换整合频率与0.35和2.5kb之间的DNA片段的同源性长度成比例。使用随机染色体1-kb片段，我们发现同源整合可以发生沿着整个染色体。我们利用滚环复制对分子内重组的刺激作用，开发了一种基因置换方案。首先将培养物保持在37 ℃以选择富含质粒整合体的细菌群体;通过温度变化至28 ℃激活整合的滚环质粒导致通过同源重组的有效质粒切除和通过质粒携带的修饰拷贝替换染色体基因。当对替换基因进行选择时，超过50%的细胞经历了替换重组。当不进行选择时，1%至40%的细胞进行置换重组。以这种方式获得染色体插入和缺失。这些结果表明，通过利用递送载体的热敏滚环性质，可以以极高的效率获得基因置换。该方法适用于多种革兰氏阳性菌。
A system for high-efficiency single- and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG+host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37-degrees-C. A nested set of L. lactis chromosomal fragments cloned onto pG+host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37-degrees-C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28-degrees-C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40% of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.