Thirty-eight C-terminal amino acids of the coupling protein TraD of the F-like conjugative resistance plasmid R1 are required and sufficient to confer binding to the substrate selector protein TraM
Thirty-eight C-terminal amino acids of the coupling protein TraD of the F-like conjugative resistance plasmid R1 are required and sufficient to confer binding to the substrate selector protein TraM
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DOI：
10.1128/jb.186.20.6999-7006.2004
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发表时间：
2004-10-01
影响因子：
3.2
通讯作者：
Koraimann, G
中科院分区：
文献类型：
--
作者：
Beranek, A;Zettl, M;Koraimann, G
偶联蛋白(CP)存在于植物、动物和人类病原体的IV型分泌系统中，是细菌结合系统中DNA转移所必需的。CP将DNA处理机器连接到交配配对形成转移设备。在这份报告中，我们提供了体外和体内的数据，证明了IncFII R1质粒转移系统的CP Trad与松弛小体蛋白TRAM特异性结合。通过重叠分析和酶联免疫吸附分析，我们发现Trad的一个截短版本，称为TraD11(DeltaN155)，与TRAM强烈相互作用。测定了TRAD11-TRAM的表观结合常数为2.6×10(7)升/摩尔。电泳迁移率改变分析表明，当该变异体与其靶DNA形成络合物时，它也与TRAM强烈结合。当从Trad的C末端额外去掉38个氨基酸时，没有观察到与TRAM的结合。TraD15由Trad的38个氨基酸组成，与TRAM结合，表明TRAM的主要相互作用区域位于Trad的这38个氨基酸中。TraD15对菌毛特异性噬菌体R17的DNA转移有显性负效应，但对噬菌体感染无显性负效应，表明TRAM-Trad相互作用对接合DNA转移有重要作用，但对噬菌体感染无影响。我们还观察到，Trad由与R1TRAM紧密相关的F因子编码。因此，我们的结果提供了证据，证明INCF质粒组内的底物选择是基于TRAM选择正确的DNA分子进行运输的能力，而不是基于CP对底物的选择。
Coupling proteins (CPs) are present in type IV secretion systems of plant, animal, and human pathogens and are essential for DNA transfer in bacterial conjugation systems. CPs connect the DNA-processing machinery to the mating pair-forming transfer apparatus. In this report we present in vitro and in vivo data that demonstrate specific binding of CP TraD of the IncFII R1 plasmid transfer system to relaxosomal protein TraM. With overlay assays and enzyme-linked immunosorbent assays we showed that a truncated version of TraD, termed TraD11 (DeltaN155), interacted strongly with TraM. The apparent TraD11-TraM association constant was determined to be 2.6 X 10(7) liters/mol. Electrophoretic mobility shift assays showed that this variant of TraD also strongly bound to TraM when it was in complex with its target DNA. When 38 amino acids were additionally removed from the C terminus of TraD, no binding to TraM was observed. TraD15, comprising the 38 amino-acid-long C terminus of TraD, bound to TraM, indicating that the main TraM interaction domain resides in these 38 amino acids of TraD. TraD15 exerted a dominant negative effect on DNA transfer but not on phage infection by pilus-specific phage R17, indicating that TraM-TraD interaction is important for conjugative DNA transfer but not for phage infection. We also observed that TraD encoded by the closely related F factor bound to TraM encoded by the R1 plasmid. Our results thus provide evidence that substrate selection within the IncF plasmid group is based on TraM's capability to select the correct DNA molecule for transport and not on substrate selection by the CP.