Mechanism of conjugal transfer in plasmid R64

IncI1质粒R64的54kb转移区包含49个基因，其中24个基因是表面配对所必需的。位于细菌毛顶端的液体交配还需要编码细菌毛的12个pil基因。R64Shufflon的多重DNA倒置选择了具有不同C-末端片段的7个PilV粘附素中的一个。1)共分离到43个编码Pilu蛋白的突变体，表明Pilu蛋白属于天冬氨酸肽酶家族。通过构建Pilu-PhoA和Pilu-LacZ融合基因，揭示了Pilu复杂的膜拓扑结构。2)R64 SFX改组序列是不对称的。检测到人工对称SFX序列之间的直接定向重组。使用对称SFX序列的重组不需要RCI C-末端片段。通过印迹实验和Biaccore分析，证实了受体细胞包膜内毒素与各种PilV粘附素C-末端片段的特异性结合。3)R64的trbAC基因不是R64动员CloDF13所必需的，提示trbAC产物的功能与F Trad相似。4)R64 ORIT操纵子由ORIT序列和nikab基因组成。从ORIT DNA和纯化蛋白构建的ORIT-nikab复合体中引入了ORIT特异性缺口。5)INCIγ质粒R621a的转移区与R64相似，但它们的托盘和exc基因结构不同。R621a和R64属于不同的排除群。构建了许多托盘突变体，以确定决定排斥特异性的区域。

The 54-kb transfer region of IncI1 plasmid R64 contains 49 genes, in which 24 genes are essential for surface matings. Additional 12 pil genes encoding thin pili are required for liquid matings, located at the tip of thin pili. Multiple DNA inversions of R64 shufflon select one of seven PilV adhesins with different C-terminal segments. 1)Forty-three mutants of pilU gene encoding prepilin peptidase were isolated, suggesting that PilU protein belongs to the aspartic acid peptidase family. Complicated membrane topology of PilU was suggested by constructing pilU-phoA and pilU-lacZ fusion genes. 2)R64 sfx shufflon recombination sequences have been shown to be asymmetric. Recombination between artificial symmetric sfx sequences in a direct orientation was detected. The Rci C-terminal segment was not required for the recombination using symmetric sfx sequences. Specific binding of lipopolysaccharide of recipient cell envelope to C-terminal segments of various PilV adhesins was demonstrated by blotting experiments and Biacore analyses. 3)R64 trbAC genes were shown to be not required for the mobilization of CloDF13 by R64, suggesting that trbAC products act the function similar to F TraD. 4)The R64 oriT operon consists of oriT sequence and nikAB genes. Introduction of oriT-specific nick was demonstrated for oriT-NikAB complex constructed from oriT DNA and purified proteins. 5)Transfer region of IncIγ plasmid R621a was found to be similar to that of R64, although the structures of their traY and exc genes are different each other. R621a and R64 were found to belong to distinct exclusion groups. Many traY mutants were constructed to identify the region determining exclusion specificity.