He Yu-Zhang, Li Xing-Ping, Miao Yuan-Yuan, Lin Jun, Sun Ruan-Yang, Wang Xiao-Pei, Guo Ya-Ya, Liao Xiao-Ping, Liu Ya-Hong, Feng Youjun, Sun Jian
National Risk Assessment Laboratory for Antimicrobial Resistance of Animal Original Bacteria, South China Agricultural University, Guangzhou, China.
Guangdong Provincial Key Laboratory of Veterinary Pharmaceutics Development and Safety Evaluation, South China Agricultural University, Guangzhou, China.
Front Microbiol. 2019 Jan 22;10:15. doi: 10.3389/fmicb.2019.00015. eCollection 2019.
The mobile colistin resistance gene is a serious threat to global human and animal health. The composite transposon Tn and its circular intermediate were proposed to be involved in the spread of but their roles remain poorly understood. To further explore the intermediates during the transposition of Tn, we engineered strains that carry an intact Tn transposon or its deletion derivatives. PCR assays were performed to detect IR-IR junctions and possible circular intermediates. We carried out transposition experiments to calculate transposition frequency. The transposition sites were characterized by whole genome sequence and ISMapper-based analyses. The presence of an intact Tn was demonstrated to be essential for the successful transposition of , although both Tn and Tn-ΔIR could form circular intermediates. The insertion sequence junction structure was observed in all constructed plasmids but the IS dimer was only formed in one construct containing an intact Tn. The average frequency of transposition in an strain possessing an intact Tn was ∼10 per transformed cell. We identified 27 integration sites for the Tn transposition event. All the transposition sites were flanked by 2 bp target duplications and preferentially occurred in AT-rich regions. These results indicate that transposition relies on the presence of an intact Tn. In addition, formation of the tandem repeat IS could represent a crucial intermediate. Taken together, the current investigations provide mechanistic insights in the transposition of .
可移动的黏菌素耐药基因对全球人类和动物健康构成严重威胁。复合转座子Tn及其环状中间体被认为与该基因的传播有关，但它们的作用仍知之甚少。为了进一步探索Tn转座过程中的中间体，我们构建了携带完整Tn转座子或其缺失衍生物的菌株。进行PCR检测以检测反向重复序列（IR）-IR连接点和可能的环状中间体。我们进行了转座实验以计算转座频率。通过全基因组测序和基于ISMapper的分析对转座位点进行了表征。结果表明，完整Tn的存在对于该转座的成功至关重要，尽管Tn和Tn-ΔIR都可以形成环状中间体。在所有构建的质粒中都观察到了插入序列连接结构，但IS二聚体仅在一个含有完整Tn的构建体中形成。在含有完整Tn的大肠杆菌菌株中，该转座的平均频率约为每转化细胞10次。我们确定了Tn转座事件的27个整合位点。所有转座位点两侧都有2 bp的靶标重复序列，并且优先发生在富含AT的区域。这些结果表明该转座依赖于完整Tn的存在。此外，串联重复IS的形成可能代表一个关键中间体。综上所述，目前的研究为该转座提供了机制上的见解。
