Wang H, Mullany P
Department of Microbiology, Eastman Dental Institute for Oral Health Care Sciences, University College London, London WC1X 8LD, United Kingdom.
J Bacteriol. 2000 Dec;182(23):6577-83. doi: 10.1128/JB.182.23.6577-6583.2000.
Tn5397 is a novel conjugative transposon, originally isolated from Clostridium difficile. This element can transfer between C. difficile strains and to and from Bacillus subtilis. It encodes a conjugation system that is very similar to that of Tn916. However, insertion and excision of Tn5397 appears to be dependent on the product of the element encoded gene tndX, a member of the large resolvase family of site-specific recombinases. To test the role of tndX, the gene was cloned and the protein was expressed in Escherichia coli. The ability of TndX to catalyze the insertion and excision of derivatives (minitransposons) of Tn5397 representing the putative circular and integrated forms, respectively, was investigated. TndX was required for both insertion and excision. Mutagenesis studies showed that some of the highly conserved amino acids at the N-terminal resolvase domain and the C-terminal nonconserved region of TndX are essential for activity. Analysis of the target site choices showed that the cloned Tn5397 targets from C. difficile and B. subtilis were still hot spots for the minitransposon insertion in E. coli.
Tn5397是一种新型接合转座子，最初从艰难梭菌中分离得到。该元件可在艰难梭菌菌株之间转移，也可在艰难梭菌与枯草芽孢杆菌之间相互转移。它编码一种与Tn916非常相似的接合系统。然而，Tn5397的插入和切除似乎依赖于元件编码基因tndX的产物，tndX是位点特异性重组酶的大解离酶家族成员。为了测试tndX的作用，该基因被克隆并在大肠杆菌中表达。研究了TndX催化分别代表假定环状和整合形式的Tn5397衍生物（微型转座子）插入和切除的能力。插入和切除都需要TndX。诱变研究表明，TndX的N端解离酶结构域和C端非保守区域的一些高度保守氨基酸对活性至关重要。对靶位点选择的分析表明，从艰难梭菌和枯草芽孢杆菌克隆的Tn5397靶标仍然是微型转座子在大肠杆菌中插入的热点。
