CONJUGATIVE MOBILIZATION OF PLASMID R1162

质粒 R1162 的接合动员

• 批准号: 6191203
• 负责人: RICHARD J MEYER
• 金额: $ 26.83万
• 依托单位: UNIVERSITY OF TEXAS AT AUSTIN
• 依托单位国家: 美国
• 财政年份: 1986
• 资助国家: 美国
• 起止时间: 1986-12-01 至 2004-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6191203
• 关键词: DNA binding protein; DNA primase; DNA replication; Escherichia coli; affinity chromatography; bacterial proteins; biochemical evolution; genetic manipulation; genetic mapping; genetic recombination; host organism interaction; microorganism conjugation; molecular cloning; mutant; nucleic acid sequence; open reading frames; plasmids; point mutation; protein biosynthesis; protein purification; protein sequence; protein transport; site directed mutagenesis; transcription factor; transposon /insertion element

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.

描述（改编自调查员的摘要）：广泛的宿主范围 质粒R1162，广泛散布在不同的革兰氏阴性细菌中 物种是通过其他人连接到细胞的 自译质粒。动员需要形成 松弛体由转移的质粒起源（orit）组成，结合了三个， 质粒编码的动员（BOB）蛋白。这两个综合物都需要 处理DNA转移的DNA，并识别搭配机器 转移矢量。松弛体的主要成分MOBA是DNA 核酸内切酶和连接酶。将确定结合Orit的蛋白质结构域 通过噬菌体显示和通过结合MOBA确定的DNA识别序列 到退化的寡核苷酸。在松弛体中，有部分 Orit DNA链的分离。发生这种情况的情况将由 表征MOBA和其他松弛体蛋白与 Orit异形电子会。遗传证据表明MOBB稳定 松弛体并通过与MOBA相互作用来促进有效的转移。身体的 这种相互作用的证据将通过共免疫沉淀获得。这 Incp-1质粒RK2的魔术装置有效地动员了R1162。这 R1162识别的蛋白质成分，质粒进入A 转移循环将通过亲和力覆盖程序确定， 或通过直接免疫沉淀。该蛋白质的相互作用结构域将 通过噬菌体显示或细菌两杂化测定法映射。共逆转 将进行研究以确定是否也认可了该领域 由RK2本身。另外，R1162中增加频率的突变 动员将被分离并测试RK2共转移的干扰。 质粒R1162和PSC101的BOB系统显然相关，相似 Orits和MOBA蛋白。但是，PSCL0L不编码R1162的同源物 蛋白质MOBB和MOBC。 PSCL0L如何独立于这些蛋白 将调查。总体而言，这些研究将提供有关如何 雷斯糖体将质粒DNA引导到链与孔进行转移。