Successful Transfer of a Model T-DNA Plasmid to E. coli Revealed Its Dependence on Recipient RecA and the Preference of VirD2 Relaxase for Eukaryotes Rather Than Bacteria as Recipients.
Successful Transfer of a Model T-DNA Plasmid to E. coli Revealed Its Dependence on Recipient RecA and the Preference of VirD2 Relaxase for Eukaryotes Rather Than Bacteria as Recipients.
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DOI：
10.3389/fmicb.2018.00895
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发表时间：
2018
影响因子：
5.2
通讯作者：
Suzuki K
中科院分区：
文献类型：
--
作者：
Ohmine Y;Kiyokawa K;Yunoki K;Yamamoto S;Moriguchi K;Suzuki K
在农杆菌介导的植物转化（AMT）中，与VirD2蛋白共价连接的单链T-DNA通过称为VirB/D4的细菌IV型分泌通道移动。这种转运系统起源于细菌的偶联质粒转运系统。松弛酶VirD2及其等效蛋白Mob分别在T-DNA转移和可移动质粒转移中起重要作用。在本研究中，我们试图通过VirB/D4装置将一个没有左边界但有右边界序列的模型T-DNA质粒和一个可移动的质粒转移到大肠杆菌、农杆菌和酵母中，比较vird2驱动的转移和mob2驱动的转移。两种类型的AMT都成功地转移到三种受体生物体上。vird2驱动的两种细菌AMT的效率低于mobi驱动的AMT。相比之下，VirD2诱导的酵母AMT比Mob诱导的AMT效率更高。从vird2驱动的AMT菌落中回收的质粒dna显示了原始的质粒结构。这些数据表明，VirD2在受体细菌细胞中保留了其大部分重要功能，但在很大程度上已适应真核生物而不是细菌。酵母的高AMT效率表明，VirD2也能有效地将ssDNA带入受体细菌细胞，但在导致细菌双链环状DNA形成的某些过程中不如Mob。本研究还发现受体recA基因显著参与vird2依赖性AMT，但仅部分参与mob依赖性AMT。两种AMT对recA基因需求的明显差异表明，VirD2在再循环以完成互补DNA合成方面比细菌中的Mob更差。
In Agrobacterium-mediated transformation (AMT) of plants, a single-strand (ss) T-DNA covalently linked with a VirD2 protein moves through a bacterial type IV secretion channel called VirB/D4. This transport system originates from conjugal plasmid transfer systems of bacteria. The relaxase VirD2 and its equivalent protein Mob play essential roles in T-DNA transfer and mobilizable plasmid transfer, respectively. In this study, we attempted to transfer a model T-DNA plasmid, which contained no left border but had a right border sequence as an origin of transfer, and a mobilizable plasmid through the VirB/D4 apparatus to Escherichia coli, Agrobacterium and yeast to compare VirD2-driven transfer with Mob-driven one. AMT was successfully achieved by both types of transfer to the three recipient organisms. VirD2-driven AMT of the two bacteria was less efficient than Mob-driven AMT. In contrast, AMT of yeast guided by VirD2 was more efficient than that by Mob. Plasmid DNAs recovered from the VirD2-driven AMT colonies showed the original plasmid structure. These data indicate that VirD2 retains most of its important functions in recipient bacterial cells, but has largely adapted to eukaryotes rather than bacteria. The high AMT efficiency of yeast suggests that VirD2 can also efficiently bring ssDNA to recipient bacterial cells but is inferior to Mob in some process leading to the formation of double-stranded circular DNA in bacteria. This study also revealed that the recipient recA gene was significantly involved in VirD2-dependent AMT, but only partially involved in Mob-dependent AMT. The apparent difference in the recA gene requirement between the two types of AMT suggests that VirD2 is worse at re-circularization to complete complementary DNA synthesis than Mob in bacteria.
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DOI：
10.3109/10409238.2012.729562
发表时间：
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影响因子：
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作者：
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DOI：
10.1016/j.bbamcr.2013.12.019
发表时间：
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影响因子：
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作者：
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