CONJUGATIVE MOBILIZATION OF PLASMID R1162

描述(改编自调查人员的摘要)：广泛的宿主范围 广泛分布于不同革兰氏阴性菌中的R1162质粒 从一个细胞转移到另一个细胞， 可自我传播的质粒。动员需要形成一种 松弛小体由一个转移质粒起点(ORIT)组成，结合三个， 质粒编码动员(MOB)蛋白。这座建筑群既需要 处理DNA以供转移，并识别 转移载体。松弛小体MOBA的主要成分是DNA 核酸内切酶和连接酶。将确定与ORIT结合的蛋白质结构域 通过噬菌体展示和通过结合MOBA确定的DNA识别序列 变成一种简并的寡核苷酸。在松弛小体内，有部分 ORIT DNA链的分离。将调查这种情况是如何发生的 MOBA和其他松弛小体蛋白与 ORIT异质双链。遗传证据表明，Mobb稳定了 松弛小体，并通过与MOBA相互作用促进有效的转移。物理 这种相互作用的证据将通过免疫共沉淀获得。这个 IncP-1载体RK2的结合器能有效地动员R1162。这个 R1162识别的蛋白质组分，是质粒进入 将通过亲和力覆盖过程来识别一轮转移， 或通过直接免疫沉淀。这种蛋白质的相互作用结构域将 通过噬菌体展示或细菌双杂交实验进行定位。共同回归 将进行研究，以确定这一领域是否也得到承认 RK2本身。此外，R1162基因的突变增加了 将分离动员并测试RK2共转移的干扰。 质粒R1162和pSC101的MOB系统明显相关，与类似的 Orits和MOBA蛋白。然而，pSCL01没有编码与R1162同源的基因 蛋白MobB和MobC。PSCL01是如何独立于这些蛋白质的 将被调查。总体而言，这些研究将使我们深入了解 松弛体会将质粒DNA引导到结合孔进行转移。

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.