Farrand S K, Slota J E, Shim J S, Kerr A
Plasmid. 1985 Mar;13(2):106-17. doi: 10.1016/0147-619x(85)90063-0.
The kanamycin-resistance transposon Tn5 was randomly introduced into pAgK84, a 47.7-kb plasmid coding for agrocin 84 production in Agrobacterium. Using such marked plasmids, pAgK84 was found to be conjugal. It could be transferred to several Agrobacterium strains including those harboring octopine- or nopaline-type Ti plasmids. Its presence has no effect on Ti plasmid functions such as opine utilization and tumorigenicity, but it does confer agrocin 84 immunity upon previously sensitive strains. The plasmid could also be conjugally transferred to a Nod+ Fix+ strain of Rhizobium meliloti. The production of agrocin 84 is expressed in all Agrobacterium and Rhizobium transconjugants tested. The agrocin plasmid could not be introduced into restrictionless Escherichia coli or Pseudomonas aeruginosa recipients by conjugation or transformation. The sites of 92 independent Tn5 insertions were mapped on pAgK84. These insertions are dispersed over the entire length of the plasmid. Analysis of the sites and effects of the Tn5 insertions has allowed us to construct a functional map of pAgK84. Forty-three of these insertions, spanning a 20-kb segment of the plasmid, abolished or greatly reduced the production of agrocin 84. The presence of two insertions within this segment having an effect on agrocin production suggests that at least three regions of the plasmid are involved in agrocin 84 biosynthesis. Fourteen of the Tn5 insertion derivatives are no longer conjugally transferable. These insertions all map to a single region of the plasmid and define about 3.5-kb as being associated with transfer functions.
卡那霉素抗性转座子Tn5被随机导入pAgK84，pAgK84是一种47.7 kb的质粒，编码农杆菌中农杆菌素84的产生。使用这种带有标记的质粒，发现pAgK84具有接合性。它可以转移到几种农杆菌菌株，包括那些携带章鱼碱型或胭脂碱型Ti质粒的菌株。它的存在对Ti质粒的功能如冠瘿碱利用和致瘤性没有影响，但它确实赋予先前敏感菌株农杆菌素84免疫性。该质粒也可以通过接合转移到苜蓿根瘤菌的一个Nod+ Fix+菌株中。在所有测试的农杆菌和根瘤菌接合子中都表达了农杆菌素84的产生。农杆菌素质粒不能通过接合或转化导入无限制的大肠杆菌或铜绿假单胞菌受体中。92个独立的Tn5插入位点被定位在pAgK84上。这些插入分散在质粒的整个长度上。对Tn5插入位点和效应的分析使我们能够构建pAgK84的功能图谱。其中43个插入跨越质粒的20 kb片段，消除或大大降低了农杆菌素84的产生。在该片段内有两个对农杆菌素产生有影响的插入表明，质粒至少有三个区域参与农杆菌素84的生物合成。14个Tn5插入衍生物不再能够通过接合转移。这些插入都定位在质粒的一个单一区域，并确定约3.5 kb与转移功能相关。
