Integration and excision of DNA at dif via XerCD-mediated site-specific recombination in Neisseria gonorrhoeae

Most strains of Neisseria gonorrhoeae, and a few strains of Neisseria meningitidis, carry the 57 kb, horizontally-acquired, gonococcal genetic island (GGI). The GGI of N. gonorrhoeae encodes a type IV secretion system, is inserted at the dif site and is flanked by a direct partial repeat, a degenerate dif. The focus of this thesis is to elucidate mechanisms of GGI integration and excision and examine factors that contribute to its excision and possible transfer.;In E. coli, the site-specific recombinases, XerC and XerD, recognize dif to resolve chromosomal dimers during replication. In N. gonorrhoeae, the presence of a conserved dif, difA, and a degenerate dif, difB, along with evidence for horizontal acquisition and absence of the GGI from some strains, suggests that the GGI may be gained or lost via site-specific recombination. Excision of the GGI from the chromosome was found to occur via both homologous recombination and XerCD-mediated site-specific recombination. Transformation of a GGI+ strain with flanking DNA deletes the GGI from the genome. DNA can be integrated into the chromosome of this DeltaGGI strain at dif. A 2 kb model island carrying difA was able to insert into the chromosome at dif. This island, flanked by two difA sites, is readily excised and lost from the genome.;GGI excision and loss via site-specific recombination requires xerD. While the GGI of an engineered strain carrying two difA copies is unstable and readily lost from the gonococcal genome, the GGI flanked by wild-type copies of dif is rarely lost. Once excised from the chromosome the GGI exists transiently as an extrachromosomal entity. Mutations in dif stabilize both the GGI and the model island in the chromosome. Accessory sequences also affect the frequency of excision at dif.;This research establishes that site-specific recombination at dif is a plausible means for GGI excision and incorporation and potentially involved in its transfer to other Neisseria. Acquisition of the GGI may have implications in pathogenesis or survival within its host. Further studies could determine whether the GGI is a mobile element and its evolutionary relevance in the pathogenic Neisseria.