Mobilization of chimeric oriT plasmids by F and R100-1:: Role of relaxosome formation in defining plasmid specificity
Mobilization of chimeric oriT plasmids by F and R100-1:: Role of relaxosome formation in defining plasmid specificity
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DOI：
10.1128/jb.182.14.4022-4027.2000
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发表时间：
2000-07-01
影响因子：
3.2
通讯作者：
Frost, LS
中科院分区：
文献类型：
--
作者：
Fekete, RA;Frost, LS
在转移起点（oriT）内的F质粒nic位点的切割需要F编码的蛋白质TraY和TraI以及宿主编码的蛋白质整合宿主因子。我们证实，F TraY，而不是F TraM，是需要在体内裂解在NIC。构建嵌合质粒，其含有完整的F或R100-1 oriT区或除traM基因外的nic、TraY和TraM结合位点的各种组合。在F或R100-1质粒存在下测定nic切割效率和动员频率。还使用转移效率测定测量了这些嵌合质粒补充F traM突变体或通过负显性影响F转移的能力。在检测到nic切割的情况下，R100-1 TraI对nic下游序列中的两个碱基差异不敏感，而F TraI对F序列具有特异性。只有当TraM能够与oriT内的同源位点结合时才能检测到质粒转移。高亲和力结合的TraY顺oriT允许检测切割在nic，但不需要有效的动员。两者合计，我们的研究结果表明，稳定的松弛体，由TraI，-M，和-Y结合oriT优先针对的转移装置（transferosome）。
Cleavage at the F plasmid nic site within the origin of transfer (oriT) requires the F-encoded proteins TraY and TraI and the host-encoded protein integration host factor in vitro. We confirm that F TraY, but not F TraM, is required for cleavage at nic in vivo. Chimeric plasmids were constructed which contained either the entire F or R100-1 oriT regions or various combinations of nic, TraY, and TraM binding sites, in addition to the traM gene. The efficiency of cleavage at nic and the frequency of mobilization were assayed in the presence of F or R100-1 plasmids. The ability of these chimeric plasmids to complement an F traM mutant or affect F transfer via negative dominance was also measured using transfer efficiency assays. In cases where cleavage at nic was detected, R100-1 TraI was not sensitive to the two-base difference in sequence immediately downstream of nic, while F TraI was specific for the F sequence. Plasmid transfer was detected only when TraM was able to bind to its cognate sites within oriT. High-affinity binding of TraY in cis to oriT allowed detection of cleavage at nic but was not required for efficient mobilization. Taken together, our results suggest that stable relaxosomes, consisting of TraI, -M, and -Y bound to oriT are preferentially targeted to the transfer apparatus (transferosome).