Molecular analysis of oriT-independent transfer in the gonococcal R-plasmids

描述（由申请方提供）：淋病奈瑟菌菌株的出现对青霉素和四环素表现出临床显著水平的耐药性，这是一个全球性问题。在共存的41 kb四环素抗性泰特（M）自传播质粒的帮助下，可将7.4 kb、5.6 kb和5.2 kb的三种编码β-内酰胺酶（R）的质粒从致病性奈瑟氏球菌动员到泌尿生殖道奈瑟氏球菌和来自生殖道和生殖道外感染部位的植物群。近年来，我们的实验室在了解淋球菌R-质粒转移的分子机制方面取得了相当大的进展，通过鉴定和表征转移（oriT）和流动酶（mob）蛋白的起源;淋球菌7.4和5.6 kb R-质粒的启动和转移所需的遗传因子。这些oriT可以被由相同β-内酰胺酶质粒编码的mob蛋白或来自自传递质粒的转移（Tra）装置的mob蛋白识别。5.2 kb R-质粒是7.4 kb R-质粒的oriT / mob缺失衍生物，其在淋球菌中的动员机制仍有待阐明。我们发现在圣胡安分离的5.2 kb R-质粒pSJ5.2可以在肠杆菌R64、N3和41 kb泰特（M）接合质粒的帮助下通过融合（共整合）被转移到大肠杆菌中。本研究的目的是探讨41 kb泰特（M）接合质粒在淋球菌中接合转移pSJ5.2的分子机制。该研究的中心假设是pSJ5.2可以在淋球菌中移动，因为它可以采用重组机制，导致与41 kb泰特（M）接合质粒共整合。将从交配试验期间产生的泰特（M）：：pSJ5.2共整合体中拯救pSJ5.2质粒，以对质粒整合体侧翼的连接进行测序，并阐明质粒重组的中间体。与转移相关的其它功能性oriT和顺式作用位点将通过体内径流DNA实验、含有pSJ5.2和泰特（M）片段的质粒构建体的合成、在E.大肠杆菌、DNA测序以及使用基因库数据库进行比较分析。首次在泰特（M）质粒中测定Tra装置和其他与转移相关的DNA位点。本研究的结果将有助于设计创新的方法来控制细菌间耐药质粒的水平传播。 公共卫生相关性：本研究的主要目的是了解耐药质粒在不同病原菌间横向转移的分子机制。 本课题组首次发现淋病奈瑟菌的5.2kb β-内酰胺酶质粒通过与大肠杆菌的多个接合质粒和泰特（M）淋球菌接合质粒共整合而被动员。这项研究将有助于设计创新的方法来控制细菌间耐药质粒的水平传播。

DESCRIPTION (provided by applicant): The emergence of strains of Neisseria gonorrhoeae that express clinically significant levels of resistance to penicillin and tetracycline is a global problem. Three beta-lactamase-encoding (R) plasmids of 7.4 kb, 5.6 kb and 5.2 kb can be mobilized from the pathogenic Neisseria to commensal Neisseria and the flora from the urogenital and extragenital sites of infection with the help of the co-resident 41 kb tetracycline- resistance, tet(M) self-transmissible plasmid. In recent years, considerable advances have been made in our laboratory in understanding the molecular machinery of transfer of the gonococcal R-plasmids through the identification and characterization of the origin of transfer (oriT) and mobilase (mob) proteins; the genetic factors required for the initiation and transfer of the gonococcal 7.4 and 5.6 kb R-plasmids. These oriTs can be recognized either by mob proteins coded by the same beta-lactamase plasmid or by mob proteins from the Transfer (Tra) apparatus of the self-transmissible plasmid. The 5.2 kb R- plasmid is an oriT / mob deletion derivative of the 7.4 kb R-plasmid whose mechanism of mobilization in the gonococci remain to be elucidated. We found that the 5.2 kb R- plasmid isolated in San Juan, pSJ5.2, can be mobilized to Escherichia coli by fusion (cointegration) with the help of the Enterobacterial R64, N3 and the 41 kb tet(M) conjugative plasmids. The objective of this project is to determine the molecular mechanism involved in the conjugal transfer of pSJ5.2 by the 41 kb tet(M) conjugative plasmid in the gonococcus. The central hypothesis of the study is that pSJ5.2 can be mobilized in the gonococcus because it can employ mechanisms of recombination leading to cointegration with the 41 kb tet(M) conjugative plasmid. The pSJ5.2 plasmid will be rescued from tet(M)::pSJ5.2 cointegrates generated during mating assays to sequence the junctions flanking the plasmid integrate and elucidate the intermediate(s) for the plasmid recombination. Other functional oriT's and cis-acting sites associated with transfer will be determined by in vivo runoff DNA experiments, synthesis of plasmid constructs containing fragments of pSJ5.2 and tet(M), complementation assays with Tra proteins from tet(M) provided in Trans during mobilization experiments in E. coli, DNA sequencing, and comparative analysis using GenBank databases. For the first time the Tra apparatus and other DNA loci related to transfer will be determined in the tet(M) plasmid. The result of this research will help to design innovative methods to control the horizontal transmission of resistance plasmids among bacteria. PUBLIC HEALTH RELEVANCE: The main goal of our project is to understand the molecular machinery of mobilization involved in the lateral transfer of resistance plasmid among different pathogenic bacteria. For the first time, our group showed that the 5.2 kb beta-lactamase plasmid of Neisseria gonorrhea is mobilized by cointegration with several conjugative plasmids of Escherichia coli and the tet(M) gonococcal conjugative plasmid. This research will help to design innovative methods to control the horizontal transmission of resistance plasmids among bacteria.