A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation
A stable shuttle vector system for efficient genetic complementation of Helicobacter pylori strains by transformation and conjugation
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DOI：
10.1007/s004380050677
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发表时间：
1998-03-01
期刊：
影响因子：
--
通讯作者：
Haas, R
中科院分区：
文献类型：
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作者：
Heuermann, D;Haas, R
构建了一种多用途质粒穿梭载体系统，用于幽门螺杆菌菌株或突变体与同源或异源克隆基因的遗传互补。单个质粒载体由最基本的遗传元素组成，包括大肠杆菌的复制起源、最初在幽门螺杆菌小隐质粒上鉴定的幽门螺杆菌特异性复制子、oriT序列和多个克隆位点。穿梭质粒pHe12携带一个氯霉素耐药盒（cat(GC)）， pHe13携带一个卡那霉素耐药基因（aphA-3）作为选择性标记；两者都在大肠杆菌和幽门螺杆菌中起作用。通过自然转化将穿梭质粒导入幽门螺杆菌P1。用pHe12和pHe13分别获得7.0 × 10（-7）和4.7 × 10（-7）个转化子的效率，并且这两种载体在幽门螺杆菌中都表现出稳定、自主的复制。当从同源幽门螺杆菌菌株中分离出用于转化的穿梭载体时，幽门螺杆菌的转化率比大肠杆菌高约100倍，这表明DNA的限制性修饰机制在质粒转化中起着至关重要的作用。有趣的是，这两种穿梭载体也可以有效地从大肠杆菌转移到不同的幽门螺杆菌受体中，pHe12的效率为每个活的幽门螺杆菌P1受体2.0 × 10（-5）个转偶联体。因此，在配偶转移过程中，DNA限制似乎大大减少或不存在。通过克隆的幽门螺杆菌recA'基因对缺乏leca的幽门螺杆菌突变体的功能互补，以及异种绿色荧光蛋白（GFP）在幽门螺杆菌中的表达，证明了该系统的普遍适用性，为今后幽门螺杆菌毒力因子的分子分析提供了重要的依据。
A versatile plasmid shuttle vector system was constructed, which is useful for genetic complementation of Helicobacter pylori strains or mutants with cloned genes of homologous or heterologous origin. The individual plasmid vectors consist of the minimal essential genetic elements, including an origin of replication for Escherichia coli, a H. pylori-specific replicon originally identified on a small cryptic H. pylori plasmid, an oriT sequence and a multiple cloning site. Shuttle plasmid pHe12 carries a chloramphenicol resistance cassette (cat(GC)) and pHe13 contains a kanamycin resistance gene (aphA-3) as the selectable marker; both are functional in E. coli and H. pylori. The shuttle plasmids were introduced into the H. pylori strain P1 by natural transformation. A efficiency of 7.0 x 10(-7) and 4.7 x 10(-7) transformants per viable recipient was achieved with pHe12 and pHe13, respectively, and both vectors showed stable, autonomous replication in N. pylori. An approximately 100-fold higher H. pylori transformation rate was obtained when the shuttle vectors for transformation were isolated from the homologous H. pylori strain, rather than E. coli, indicating that DNA restriction and modification mechanisms play a crucial role in plasmid transformation. Interestingly, both shuttle vectors could also be mobilized efficiently from E. coli into different H. pylori recipients, with pHe12 showing an efficiency of 2.0 x 10(-5) transconjugants per viable H. pylori P1 recipient. Thus, DNA restriction seems to be strongly reduced or absent during conjugal transfer. The functional complementation of a lecA-deficient H. pylori mutant by the cloned H. pylori recA' gene, and the expression of the heterologous green fluorescent protein (GFP) in H. pylori demonstrate the general usefulness of this system, which will significantly facilitate the molecular analysis of H. pylori virulence factors in the future.