Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation.
Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation.
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脆弱拟杆菌可动员转座子 Tn5520 需要 71 个碱基对的转移序列起点和单个动员蛋白，以便在接合过程中形成松弛体。
DOI：
10.1111/j.1365-2958.2005.04934.x
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发表时间：
2006
期刊：
影响因子：
--
通讯作者：
Hecht,DavidW
中科院分区：
文献类型：
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作者：
Vedantam,Gayatri;Knopf,Sarah;Hecht,DavidW
tn5520是已知最小的细菌可调动转座子，从耐抗生素的脆弱拟杆菌临床分离物中分离出来。当在trans中提供偶联装置时，tn5520在两个拟杆菌中被有效地动员（转移）。andEscherichia杆菌。只有两个基因存在于Tn5520上；一个编码整合酶，另一个编码多功能动员（Mob）蛋白BmpH。bmh对Tn5520mobility至关重要。本研究的重点是确定共轭转移（oriT）的tn5520起源，并研究BmpH - oriTbinding。我们将功能性的Tn5520序列定位在bmphgene上游的一个71 bp的序列上。携带这个最小的71个bporit的质粒载体以与完整的Tn5520相同的频率被动员。最小值包含一个17bp的反向重复（IR）序列。我们构建并测试了多个IR突变体，并表明IR在其整体上对动员至关重要。在minimaloriT中也发现了一个缺口位点序列（5 ‘‐GCTAC‐3 ’）；该序列类似于革兰氏阳性质粒中的缺口位点。我们进一步发现，缺口位点序列中高度保守的GC二核苷酸的突变完全消除了动员。我们还纯化了BmpH，并在电泳迁移率转移试验中表明它特异性地结合了Tn5520 oriTfragment。我们还发现了对动员至关重要的minimalite中的非缺口位点序列。我们假设基于转座子的单一Mob蛋白系统可能有助于有效地传播拟杆菌的基因。因为松弛体形成所需的DNA加工蛋白较少。
Tn5520is the smallest known bacterial mobilizable transposon and was isolated from an antibiotic resistantBacteroides fragilisclinical isolate. When a conjugation apparatus is providedin trans, Tn5520is mobilized (transferred) efficiently within, and from, bothBacteroidesspp. andEscherichia coli.Only two genes are present on Tn5520; one encodes an integrase, and the other a multifunctional mobilization (Mob) protein BmpH. BmpH is essential for Tn5520mobility. The focus of this study was to identify the Tn5520origin of conjugative transfer (oriT) and to study BmpH‐oriTbinding. We delimited the functional Tn5520 oriTto a 71 bp sequence upstream of thebmpHgene. A plasmid vector harbouring this minimal 71 bporiTwas mobilized at the same frequency as that of intact Tn5520. The minimaloriTcontains one 17 bp inverted repeat (IR) sequence. We constructed and tested multiple IR mutants and showed that the IR was essential in its entirety for mobilization. A nick site sequence (5′‐GCTAC‐3′) was also identified within the minimaloriT; this sequence resembled nick sites found in plasmids of Gram positive origin. We further showed that mutation of a highly conserved GC dinucleotide in the nick site sequence completely abolished mobilization. We also purified BmpH and showed that it specifically bound a Tn5520 oriTfragment in electrophoretic mobility shift assays. We also identified non‐nick site sequences within the minimaloriTthat were essential for mobilization. We hypothesize that transposon‐based single Mob protein systems may contribute to efficient gene dissemination fromBacteroidesspp., because fewer DNA processing proteins are required for relaxosome formation.