CONJUGATIVE MOBILIZATION OF PLASMID R1162

描述（改编自研究者摘要）：广泛的宿主范围 质粒R1162，广泛分布于不同革兰氏阴性菌中 物种，是共轭转移从细胞到细胞的其他， 自我传播质粒动员需要形成一个 松弛体由质粒转移起点（oriT）组成，与三个， 质粒编码的移动（Mob）蛋白。这座建筑物既需要 处理DNA转移，并认识到配偶的机器， 转移载体松弛体的主要成分MobA是DNA， 核酸内切酶和连接酶。将鉴定结合oriT的蛋白质结构域 通过噬菌体展示，并通过结合MobA确定DNA识别序列 简并寡核苷酸。在松弛体内，有部分 oriT DNA链的分离。这是如何发生的，将由 表征MobA和其他松弛体蛋白与 oriT异源双链体。遗传证据表明，MobB稳定了 松弛体并通过与MobA相互作用促进有效转移。物理 这种相互作用的证据将通过共免疫沉淀获得。的 IncP-1质粒RK 2的接合器有效地动员R1162。的 由R1162识别的蛋白质组分，并且是质粒进入 传送轮将通过亲和覆盖过程来识别， 或通过直接免疫沉淀。这种蛋白质的相互作用结构域将 通过噬菌体展示或通过细菌双杂交测定来定位。一个共同逆转 将进行研究，以确定这一领域是否也得到承认 RK 2本身此外，R1162中的突变增加了 将分离动员并检测RK 2共转移的干扰。 质粒R1162和pSC 101的mob系统明显相关，具有相似的 oriTs和MobA蛋白。然而，pSC 101不编码R1162的同源物 蛋白质MobB和MobC。pSCl 01如何变得独立于这些蛋白质 将被调查。总的来说，这些研究将深入了解 松弛体将质粒DNA引导至接合孔进行转移。

DESCRIPTION (adapted from the investigator's abstract): The broad host-range plasmid R1162, widely disseminated among different Gram-negative bacterial species, is conjugally transferred from cell to cell by other, self-transmissible plasmids. Mobilization requires the formation of a relaxosome comprised of a plasmid origin of transfer (oriT), bound to three, plasmid-encoded mobilization (Mob) proteins. This complex is required both to process the DNA for transfer, and to recognize the conjugal machinery of the transferring vector. The principal component of the relaxosome, MobA, is a DNA endonuclease and ligase. The protein domain that binds oriT will be identified by phage display, and the DNA recognition sequence determined by binding MobA to a degenerate oligonucleotide. Within the relaxosome, there is partial separation of the oriT DNA strands. How this occurs will be investigated by characterizing the interaction of MobA and the other relaxosome proteins with oriT heteroduplexes. Genetic evidence indicates that MobB stabilizes the relaxosome and promotes efficient transfer by interacting with MobA. Physical evidence for this interaction will be obtained by coimmunoprecipitation. The conjugal apparatus of the IncP-1 plasmid RK2 efficiently mobilizes R1162. The protein component recognized by R1162 and required for the plasmid to enter a round of transfer will be identified either by an affinity overlay procedure, or by direct immunoprecipitation. The interacting domain of this protein will be mapped by phage display or by a bacterial two-hybrid assay. A co-reversion study will be carried out to establish whether this domain is also recognized by RK2 itself. In addition, mutations in R1162 that increase the frequency of mobilization will be isolated and tested for interference of RK2 cotransfer. The mob systems of plasmids R1162 and pSC101 are clearly related, with similar oriTs and MobA proteins. However, pSCl0l encodes no homologs to the R1162 proteins MobB and MobC. How pSCl0l has become independent of these proteins will investigated. Overall, these studies will provide insight into how the relaxosome directs plasmid DNA to the conjugal pore for transfer.