Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity.
Mutational analysis of essential IncP alpha plasmid transfer genes traF and traG and involvement of traF in phage sensitivity.
复制标题
必需 IncP α 质粒转移基因 traF 和 traG 的突变分析以及 traF 与噬菌体敏感性的关系。
DOI：
10.1128/jb.174.20.6666-6673.1992
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发表时间：
1992
影响因子：
3.2
通讯作者：
Guiney,DG
中科院分区：
文献类型：
--
作者：
Waters,VL;Strack,B;Pansegrau,W;Lanka,E;Guiney,DG
虽然广泛的宿主范围IncP质粒可以在不同的革兰氏阴性细菌中进行植物性复制，但穿梭载体系统的发展已经确定IncP质粒接合转移的宿主范围大于维持IncP复制子的细菌范围。为了理解IncP质粒接合和IncP接合与根癌农杆菌T-DNA转移到植物之间的联系，在IncP α质粒RK 2的较大转移区（Tra 1）中产生了两组突变体。选择突变策略以最小化转录极性效应。突变体Tra 1克隆进行映射，测序，并处理，以重建49.5 kb Tra 2含有质粒衍生物，以测定转移活性和IncP质粒特异性噬菌体的敏感性。聚焦于大肠杆菌中traF和traG基因产物的活性，我们发现traF中的突变废除了转移活性并使宿主细胞具有噬菌体抗性，traG中的突变废除了转移活性但对噬菌体敏感性没有影响。这些突变体衍生物与相应的反式作用克隆携带traF或traG的互补恢复转移活性，并在traF突变体的情况下，宿主细胞的噬菌体敏感性。我们的结论是，在E.大肠杆菌中，TraF和TraG都是IncP质粒转移所必需的，TraF是供体特异性噬菌体敏感性所必需的（但不是足够的），测序数据表明TraF和TraG都是跨膜的。
Although the broad-host-range IncP plasmids can vegetatively replicate in diverse gram-negative bacteria, the development of shuttle vector systems has established that the host range for IncP plasmid conjugative transfer is greater than the range of bacteria that sustain IncP replicons. Towards understanding IncP plasmid conjugation and the connection between IncP conjugation and Agrobacterium tumefaciens T-DNA transfer to plants, two sets of mutants were generated in the larger transfer region (Tra1) of the IncP alpha plasmid RK2. Mutagenesis strategies were chosen to minimize transcriptional polar effects. Mutant Tra1 clones were mapped, sequenced, and processed to reconstruct 49.5-kb Tra2-containing plasmid derivatives in order to assay for transfer activity and IncP plasmid-specific phage sensitivity. Focusing on the activities of the gene products of traF and traG in Escherichia coli, we found that mutations in traF abolished transfer activity and rendered the host cells phage resistant and mutations in traG abolished transfer activity but had no effect on phage sensitivity. Complementation of these mutant derivatives with corresponding trans-acting clones carrying traF or traG restored transfer activity and, in the case of the traF mutant, the phage sensitivity of the host cell. We conclude that in E. coli, both TraF and TraG are essential for IncP plasmid transfer and that TraF is necessary (but not sufficient) for donor-specific phage sensitivity, and sequencing data suggest that both TraF and TraG are membrane spanning.