GLAVAHCS, Los Angeles, CA, USA; UCLA School of Medicine, Los Angeles, CA 90073, USA.
FEMS Microbiol Lett. 2012 Aug;333(2):94-100. doi: 10.1111/j.1574-6968.2012.02602.x. Epub 2012 Jun 18.
Genetic analysis of Bacteroides fragilis (BF) is hindered because of the lack of efficient transposon mutagenesis methods. Here, we describe a simple method for transposon mutagenesis using EZ::TN5, a commercially available system that we optimized for use in BF638R. The modified EZ::TN5 transposon contains an Escherichia coli conditional origin of replication, a kanamycin resistance gene for E. coli, an erythromycin resistance gene for BF , and 19 basepair transposase recognition sequences on either ends. Electroporation of the transposome (transposon-transposase complex) into BF638R yielded 3.2 ± 0.35 × 10(3) CFU μg(-1) of transposon DNA. Modification of the transposon by the BF638R restriction/modification system increased transposition efficiency sixfold. Electroporation of the EZ::TN5 transposome results in a single-copy insertion of the transposon evenly distributed across the genome of BF638R and can be used to construct a BF638R transposon library. The transposon was also effective in mutating a BF clinical isolate and a strain of the related species, Bacteroides thetaiotaomicron. The EZ::TN5-based mutagenesis described here is more efficient than other transposon mutagenesis approaches previously reported for BF.
由于缺乏有效的转座子诱变方法，脆弱拟杆菌（BF）的遗传分析受到阻碍。在这里，我们描述了一种使用 EZ::TN5 进行转座子诱变的简单方法，我们对其进行了优化，使其可用于 BF638R。修饰后的 EZ::TN5 转座子包含大肠杆菌条件复制起点、大肠杆菌的卡那霉素抗性基因、BF 的红霉素抗性基因，以及两端的 19 个碱基转座酶识别序列。将转座体（转座子-转座酶复合物）电穿孔到 BF638R 中，得到 3.2 ± 0.35×10(3)CFU μg(-1)的转座子 DNA。BF638R 的限制/修饰系统对转座子的修饰将转座效率提高了六倍。EZ::TN5 转座体的电穿孔导致转座子在 BF638R 基因组中均匀分布的单拷贝插入，可用于构建 BF638R 转座子文库。该转座子在突变 BF 临床分离株和相关物种拟杆菌（Bacteroides thetaiotaomicron）菌株方面也很有效。与之前报道的用于 BF 的其他转座子诱变方法相比，这里描述的基于 EZ::TN5 的诱变方法更有效。
