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

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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.