CONSTRUCTION OF AN EAE DELETION MUTANT OF ENTEROPATHOGENIC ESCHERICHIA-COLI BY USING A POSITIVE-SELECTION SUICIDE VECTOR
CONSTRUCTION OF AN EAE DELETION MUTANT OF ENTEROPATHOGENIC ESCHERICHIA-COLI BY USING A POSITIVE-SELECTION SUICIDE VECTOR
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DOI：
10.1128/iai.59.12.4310-4317.1991
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发表时间：
1991-12-01
影响因子：
3.1
通讯作者：
KAPER, JB
中科院分区：
文献类型：
--
作者：
DONNENBERG, MS;KAPER, JB
肠致病性大肠杆菌（EPEC）的特征是能够附着在上皮细胞上，清除微绒毛表面，破坏细胞骨架。最近，人们描述了这种现象所必需的基因eae（A. e. Jerse，J. Yu，B. D.高个子，还有J. B。Kaper，Proc. Natl. Acad. sci. USA 87：7839 - 7843，1990）。我们报告使用一种新的自杀载体含有pir依赖的R6K复制子和枯草芽孢杆菌的sacB基因构建EPEC的eae缺失突变体。该系统能够针对载体序列的丢失进行正选择。所得的突变体，CVD206，是从野生型菌株，除了损失的94 kDa的外膜蛋白和附着和擦除能力的区别。在质粒上重新引入eae基因后，94-kDa外膜蛋白和附着和擦除能力都得到恢复。这些结果证实了eae基因在EPEC的附着和消失活性中的作用，并建立了用于构建缺失突变的新系统的实用性。
The ability to attach to epithelial cells, efface the microvillus surface, and disrupt the underlying cytoskeleton is characteristic of enteropathogenic Escherichia coli (EPEC). Recently, eae, a gene necessary for this phenomenon, was described (A. E. Jerse, J. Yu, B. D. Tall, and J. B. Kaper, Proc. Natl. Acad. Sci. USA 87:7839-7843, 1990). We report the use of a novel suicide vector containing the pir-dependent R6K replicon and the sacB gene of Bacillus subtilis to construct an eae deletion mutant of EPEC. This system enables positive selection for the loss of vector sequences. The resulting mutant, CVD206, is indistinguishable from the wild-type strain except for the loss of a 94-kDa outer membrane protein and attaching and effacing ability. Both the 94-kDa outer membrane protein and attaching and effacing ability are restored upon reintroduction of the eae gene on a plasmid. These results confirm the role of the eae gene in the attaching and effacing activity of EPEC and establish the utility of a new system for the construction of deletion mutations.