Sprinzl M, Geider K
Max-Planck-Institut für medizinische Forschung, Abteilung Molekulare Biologie, Heidelberg, Federal Republic of Germany.
J Gen Microbiol. 1988 Feb;134(2):413-24. doi: 10.1099/00221287-134-2-413.
We have screened strains of Agrobacterium tumefaciens for spontaneous mutants showing constitutive transfer of the nopaline Ti plasmid pTiC58 during conjugation. The Ti plasmid derivatives obtained could be transferred not only to A. tumefaciens but also to E. coli cells. The Ti plasmid cannot survive as a freely replicating plasmid in E. coli, but it can occasionally integrate into the E. coli chromosome. However, insertion in tandem of plasmids carrying fd replication origins (pfd plasmids) into the T-DNA provides an indicator for all transfer events into E. coli cells, providing fd gene 2 protein is present in these cells. This viral protein causes the excision of one copy of the pfd plasmid and allows its propagation in the host cell. By using this specially designed Ti plasmid, which was also made constitutive in transfer functions, we found plasmid exchange among A. tumefaciens strains and between A. tumefaciens and E. coli cells to be equally efficient. A Ti plasmid with repressed transfer functions was transferred to E. coli with a rate similar to the low frequency at which it was transferred to A. tumefaciens. The expression of transfer functions of plasmid RP4 either in A. tumefaciens or in E. coli did not increase the transfer of the Ti plasmid into E. coli cells, nor did the addition of acetosyringone, an inducer of T-DNA transfer to plant cells. The results show that A. tumefaciens can transfer the Ti plasmid to E. coli with the same efficiency as within its own species. Conjugational transmission of extrachromosomal DNA like the narrow-host-range Ti plasmid may often not only occur among partners allowing propagation of the plasmid, but also on a 'try-all' basis including hosts which do not replicate the transferred DNA.
我们筛选了根癌土壤杆菌菌株，以寻找在接合过程中显示胭脂碱型Ti质粒pTiC58组成型转移的自发突变体。获得的Ti质粒衍生物不仅可以转移到根癌土壤杆菌中，还可以转移到大肠杆菌细胞中。Ti质粒不能作为自由复制的质粒在大肠杆菌中存活，但它偶尔可以整合到大肠杆菌染色体中。然而，将携带fd复制起点的质粒（pfd质粒）串联插入T-DNA中，为所有进入大肠杆菌细胞的转移事件提供了一个指示，前提是这些细胞中存在fd基因2蛋白。这种病毒蛋白会导致一份pfd质粒的切除，并使其在宿主细胞中繁殖。通过使用这种经过特殊设计且转移功能也呈组成型的Ti质粒，我们发现根癌土壤杆菌菌株之间以及根癌土壤杆菌与大肠杆菌细胞之间的质粒交换效率相同。一个转移功能受到抑制的Ti质粒以与它转移到根癌土壤杆菌的低频相似的速率转移到大肠杆菌中。质粒RP4的转移功能在根癌土壤杆菌或大肠杆菌中的表达，均未增加Ti质粒向大肠杆菌细胞的转移，向植物细胞转移T-DNA的诱导剂乙酰丁香酮的添加也没有这种作用。结果表明，根癌土壤杆菌可以将Ti质粒转移到大肠杆菌中，其效率与在自身菌属内相同。像窄宿主范围的Ti质粒这样的染色体外DNA的接合传递，可能不仅经常发生在允许质粒繁殖的伙伴之间，而且也会在包括不能复制转移DNA的宿主在内的“全面尝试”基础上发生。
