Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system
Effective removal of a range of Ti/Ri plasmids using a pBBR1-type vector having a repABC operon and a lux reporter system
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使用具有repABC操纵子和lux报告系统的pBBR1型载体有效去除一系列Ti/Ri质粒
DOI：
10.1007/s00253-017-8721-7
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发表时间：
2018
影响因子：
5
通讯作者：
Suzuki Katsunori
中科院分区：
文献类型：
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作者：
Yamamoto Shinji;Sakai Ayako;Agustina Vita;Moriguchi Kazuki;Suzuki Katsunori
致病性农杆菌菌株的Ti和Ri质粒由arepAB Coperon的功能稳定维持，并被分为4个不亲和组，即inRh 1、inRh 2、inRh 3和inRh 4。从它们的细菌细胞中去除这些质粒是确定菌株特异性毒力特征和构建用于转化的菌株的重要步骤。在这里，我们开发了两个强大的工具来改进这个过程。我们首先建立了一个报告系统，通过使用来自哈氏弧菌的Ti 2小RNA和luxAB的乙酰丁香酮（AS）诱导型启动子来检测细胞中Ti/Ri质粒的存在和不存在。该系统通过使携带质粒的细胞集落响应AS发光来区分Ti/Ri无质粒细胞集落。然后，我们构建了新的“Ti/Ri驱逐质粒”，每个质粒携带来自广泛宿主范围pBBR 1载体中自杀质粒pK 18 mobsacBand中的incRh 1、incRh 2、incRh 3和incRh 4组的四种Ti/Ri质粒之一的arepABC。将新的驱逐质粒导入含有相应Ti/Ri质粒的农杆菌细胞中，导致每个inRh组中的无Ti/Ri质粒细胞。具有pBBR 1骨架的质粒比具有pK 18 mobsacB骨架的质粒更有效地清除Ti/Ri。此外，高度稳定的隐蔽质粒pAtC 58 inA.通过导入含有pAtC 58的重组质粒pABC的pBBR 1载体，有效地清除了tumefaciensC 58。这些结果表明pBBR 1-repABC质粒组是去除稳定根瘤菌质粒的有力工具。
Ti and Ri plasmids of pathogenicAgrobacteriumstrains are stably maintained by the function of arepABCoperon and have been classified into four incompatibility groups, namely, incRh1, incRh2, incRh3, and incRh4. Removal of these plasmids from their bacterial cells is an important step in determining strain-specific virulence characteristics and to construct strains useful for transformation. Here, we developed two powerful tools to improve this process. We first established a reporter system to detect the presence and absence of Ti/Ri plasmids in cells by using an acetosyringone (AS)-inducible promoter of theTi2small RNA andluxABfromVibrio harveyi. This system distinguished a Ti/Ri plasmid-free cell colony among plasmid-harboring cell colonies by causing the latter colonies to emit light in response to AS. We then constructed new “Ti/Ri eviction plasmids,” each of which carries arepABCfrom one of four Ti/Ri plasmids that belonged to incRh1, incRh2, incRh3, and incRh4 groups in the suicidal plasmid pK18mobsacBand in a broad-host-range pBBR1 vector. Introduction of the new eviction plasmids intoAgrobacteriumcells harboring the corresponding Ti/Ri plasmids led to Ti/Ri plasmid-free cells in every incRh group. The Ti/Ri eviction was more effective by plasmids with the pBBR1 backbone than by those with the pK18mobsacBbackbone. Furthermore, the highly stable cryptic plasmid pAtC58 inA. tumefaciensC58 was effectively evicted by the introduction of a pBBR1 vector containing therepABCof pAtC58. These results indicate that the set of pBBR1-repABCplasmids is a powerful tool for the removal of stable rhizobial plasmids.