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

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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.