Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes
Single-crossover integration in the Lactobacillus sake chromosome and insertional inactivation of the ptsI and lacL genes
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DOI：
10.1128/aem.63.6.2117-2123.1997
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发表时间：
1997-06-01
影响因子：
4.4
通讯作者：
MorelDeville, F
中科院分区：
文献类型：
--
作者：
Leloup, L;Ehrlich, SD;MorelDeville, F
研究了在乳酸乳杆菌中的单跨同源集成。进行了整合，无复制的递送矢量PRV300。该载体由用于在大肠杆菌和红霉素耐药性标记中传播的pbluescript sk-sk-复制子组成。将L.清晰的23K范围内的随机染色体DNA片段插入了PRV300中。所得的质粒能够通过同源重组作为单个副本整合到染色体中，并保持稳定。单个交叉积分频率与0.3至1.2 kb之间的同源性程度对数成正比，并达到1.4 x 10（3）整合体/mu g DNA的最大值。我们使用这种整合策略使PTSI基因失活，编码磷酸烯醇丙酮酸的酶：碳水化合物磷酸转移酶系统和LACL基因，这是功能性P-乳糖苷酶合成所需的两个基因之一。结果表明，我们的方法促进了清酒的遗传分析。
Single-crossover homologous integration in Lactabacillus sake was studied. Integration was conducted,vith nonreplicative delivery vector pRV300. This vector is composed of a pBluescript SK- replicon for propagation in Escherichia coli and an erythromycin resistance marker. Random chromosomal DNA fragments of L. sake 23K ranging between 0.3 and 3.4 kb were inserted into pRV300. The resulting plasmids were able to integrate into the chromosome by homologous recombination as single copies and were maintained stably. The single cross-over integration frequency was logarithmically proportional to the extent of homology between 0.3 and 1.2 kb and reached a maximum value of 1.4 x 10(3) integrants/mu g of DNA. We used this integration strategy to inactivate the ptsI gene, encoding enzyme I of the phosphoenolpyruvate:carbohydrate phosphotransferase system, and the lacL gene, which is one of the two genes required for the synthesis of a functional P-galactosidase. The results indicated that our method facilitates genetic analysis of L. sake.