Molecular Analysis of Multiple otiTs in the Gonococcal Mobilizable R-Plasmids

质粒在细菌病原体中的移动越来越重要 显然，多重耐药细菌，如淋病奈瑟菌，正在以惊人的速度出现。在淋球菌中已鉴定出7.4、5.6和5.2Kb的三种R质粒。7.4和5.6 Kb R质粒含有1.9 Kb动员盒，其具有mobA、mobC和具有功能性oriT的168 bp基因间区域。5.2 Kb R质粒缺少该盒，但可被41 Kb TetM和R6 K接合质粒移动。因此，我们的假设是，淋球菌R-质粒的混杂是由于存在两个或两个以上的转移来源，其相关的Mob蛋白的分子相互作用触发不同家族的接合质粒的质粒动员。我们建议通过序列缺失和定点突变来鉴定基因间区的功能性oriT。一个强大的选择测定动员将开发使用体内互补的MobA蛋白提供的共驻留重组质粒。对mobA和mobC基因的启动子序列的遗传分析将确定在动员转移期间相邻的Mob蛋白的转录调节作用。当5.2 Kb R质粒在接合过程中被移动时，其在1.9 Kb盒外部使用不同的oriT。将通过使用R6 K和41 Kb TetM接合质粒的互补测定来鉴定与5.2 Kb R质粒的移动相关的第二功能性oriT和反式作用蛋白。这些接合质粒将提供识别第二转移起点的外源Mob蛋白。新的独特的oriT和相关的Mob蛋白的生物学特性将通过测序、功能表征和使用基因库数据库的比较分析来确定。本研究的结果将有助于控制水平抗生素抗性基因传播的创新策略的发展，由于R-质粒的收购和多重耐药的出现在N。淋病和其他致病菌。

The importance of plasmid mobilization among bacterial pathogens is becoming increasingly apparent as multidrug resisistant bacteria, like Neisseria gonorrhoeae, are emerging at an alarming rate. Three R-plasmids of 7.4, 5.6 and 5.2 Kb have been identified in gonococcus. The 7.4 and 5.6 Kb R-plasmids contain a 1.9 Kb mobilization cassette with mobA, mobC and a 168 bp intergenic region with a functional oriT. The 5.2 Kb R-plasmid lacks this cassette but can be mobilized by the 41 Kb TetM and R6K conjugative plasmids. Therefore, our hypothesis is that the promiscuity of the gonococcal R-plasmids is due to the existence of two or more origins of transfer, and the molecular interactions with its associated Mob proteins trigger plasmid mobilization by different families of conjugative plasmids. We propose to identify the functional oriT in the intergenic region by sequence deletion and site-directed mutagenesis. A powerful selection assay for mobilization will be developed using in vivo complementation with MobA protein provided from a co-resident recombinant plasmid. Genetic analysis of the promoter sequence for mobA and mobC genes will establish the role of transcriptional regulation by adjacent Mob proteins during the mobilization transfer. The 5.2 Kb R-plasmid uses a different oriT outside from the 1.9 Kb cassette, when it is mobilized during conjugation. This second functional oriT and trans-acting proteins associated with the mobilization of the 5.2 Kb R-plasmids will be identified by complementation assays using the R6K and the 41 Kb TetM conjugative plasmids. These conjugative plasmids will provide the exogenous Mob proteins that recognize the second origin of transfer. The biological properties of the new distinct oriT and related Mob proteins will be determined by sequencing, function characterization and comparative analysis using Gene Bank databases. The result of this research will contribute to the development of innovative strategies for control of horizontal antibiotic resistance gene transmission due to R-plasmid acquisition and the emergence of multiresistance in N. gonorrhoeae and other pathogenic bacteria.