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

描述（由申请人提供）：对青霉素和四环素表现出临床显著耐药水平的淋病奈瑟菌菌株的出现是一个全球性问题。3个编码β -内酰胺酶(R)的质粒分别为7.4 kb、5.6 kb和5.2 kb，在共存的41 kb的四环素耐药tet(M)自传质粒的帮助下，可从致病性奈瑟菌转移到共生奈瑟菌和泌尿生殖道和生殖道外感染部位的菌群。近年来，通过对转移（oriT）和动员酶（mob）蛋白来源的鉴定和表征，本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；7.4 kb和5.6 kb淋球菌r质粒起始和转移所需的遗传因素。这些oriTs既可以被同一内酰胺酶质粒编码的群蛋白识别，也可以被自传递质粒的转移（Tra）装置中的群蛋白识别。5.2 kb的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尚待阐明。我们发现，在pSJ5.2圣胡安分离到的5.2 kb R-质粒可以与Enterobacterial R64， N3和41 kb tet(M)共轭质粒融合（协整）到大肠杆菌中。本项目的目的是确定41 kb tet(M)共轭质粒在淋球菌中偶联转移pSJ5.2的分子机制。本研究的中心假设是pSJ5.2可以在淋球菌中被动员，因为它可以利用重组机制导致与41 kb tet(M)共轭质粒协整。pSJ5.2质粒将从配对试验中产生的tet(M)::pSJ5.2协整体中分离出来，对质粒整合体两侧的连接进行测序，并阐明质粒重组的中间产物。其他与转移相关的功能位点和顺式作用位点将通过体内径流DNA实验、含有pSJ5.2和tet(M)片段的质粒构建体的合成、与Trans在大肠杆菌动员实验中提供的tet(M)的Tra蛋白的互补分析、DNA测序和使用GenBank数据库的比较分析来确定。这是第一次在tet(M)质粒中确定Tra装置和其他与转移相关的DNA位点。本研究结果将有助于设计创新的方法来控制耐药质粒在细菌之间的水平传播。

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.