Farrand S K, Hwang I, Cook D M
Department of Crop Sciences, University of Illinois at Urbana-Champaign, 61801, USA.
J Bacteriol. 1996 Jul;178(14):4233-47. doi: 10.1128/jb.178.14.4233-4247.1996.
The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.
根癌土壤杆菌的Ti质粒编码两个转移系统。一个介导T-DNA从细菌转移至植物细胞，而另一个负责整个Ti质粒从一个细菌到另一个细菌的接合转移。负责接合转移的决定因素定位于胭脂碱型Ti质粒pTiC58的两个区域，即tra和trb。通过使用Tn3HoHo1进行转座子诱变，我们将tra决定因素定位到一个8.5kb的区域，该区域也包含oriT区域。转座子插入形成的与lacZ的融合表明该区域表达为两个反向转录的单元。我们确定了Ti质粒一个8755bp区域的完整核苷酸序列，该区域包含定义tra的转座子插入。该区域包含六个可识别的基因，组织为两个单元，可从包含oriT位点的258bp基因间区域反向转录。一个单元编码traA、traF和traB，而第二个单元编码traC、traD和traG。位于两组基因下游的报告基因插入不影响接合，但可表达，这表明这两个单元编码在测试条件下不参与转移的其他基因。预测大小的蛋白质可从traA、traC、traD和traG表达。几个Ti质粒tra基因的产物与其他接合系统的产物相关。从traA表达的127kDa蛋白质包含与RSF1O1O的MobA以及质粒F的TraI解旋酶结构域相关的结构域。traF的翻译产物与RP4的TraF相关，traG的翻译产物与RP4的TraG以及Ti质粒T-DNA转移系统的VirD4相关。遗传分析表明，至少traG和traF对于接合转移是必需的，而序列分析预测traA也编码一种必需功能。traB虽然不是必需的，但对于最大转移频率是必需的。序列相关性模式表明，oriT和traA编码的预测同源位点特异性内切核酸酶与RSF10l0和质粒F的转移系统具有相同的谱系，而编码其他必需tra功能的Ti质粒基因与RP4接合系统的基因具有共同的祖先。
