XerCD-mediated site-specific recombination leads to loss of the 57-kilobase gonococcal genetic island.

Most strains of Neisseria gonorrhoeae carry the 57-kb gonococcal genetic island (GGI), as do a few strains of Neisseria meningitidis. The GGI is inserted into the chromosome at the dif site (difA) and is flanked by a partial repeat of the dif site (difB). Since dif is a sequence recognized by the site-specific recombinases XerC and XerD and the GGI shows evidence of horizontal acquisition, we hypothesized that the GGI may be acquired or lost by XerCD-mediated site-specific recombination. We show that while the GGI flanked by wild-type dif sites, difA and difB, is not readily lost from the gonococcal chromosome, the substitution of difB with another copy of difA allows the frequent excision and loss of the GGI. In mutants carrying two difA sites (difA(+) difA(+)), the GGI can be detected as an extrachromosomal circle that exists transiently. A mutation of xerD diminished GGI excision from the chromosome of a difA(+) difA(+) strain, while mutations in recA or type IV secretion genes had no effect on the loss of the GGI. These data indicate that the GGI is maintained by the replication of the chromosome and that GGI excision and loss are dependent upon the dif sequence and xerD. The detection of a circular form of the GGI in a wild-type strain suggests that GGI excision may occur naturally and could function to facilitate GGI transfer. These data suggest a model of GGI excision and loss explaining the absence of the GGI from some gonococcal strains and the maintenance of variant GGIs in some gonococcal and meningococcal isolates.

Type IV secretion machinery promotes ton-independent intracellular survival of Neisseria gonorrhoeae within cervical epithelial cells.

Survival of Neisseria gonorrhoeae within host epithelial cells is expected to be important in the pathogenesis of gonococcal disease. We previously demonstrated that strain FA1090 derives iron from a host cell in a process that requires the Ton complex and a putative TonB-dependent transporter, TdfF. FA1090, however, lacks the gonococcal genetic island (GGI) that is present in the majority of strains. The GGI in strain MS11 has been partially characterized, and it encodes a type IV secretion system (T4SS) involved in DNA release. In this study we investigated the role of iron acquisition and GGI-encoded gene products in gonococcal survival within cervical epithelial cells. We demonstrated that intracellular survival of MS11 was dependent on acquisition of iron from the host cell, but unlike the findings for FA1090, expression of the Ton complex was not required. Survival was not dependent on a putative TonB-like protein encoded in the GGI but instead was directly linked to T4SS structural components in a manner independent of the ability to release or internalize DNA. These data suggest that expression of selected GGI-encoded open reading frames confers an advantage during cervical cell infection. This study provides the first link between expression of the T4SS apparatus and intracellular survival of gonococci.

Neisseria gonorrhoeae secretes chromosomal DNA via a novel type IV secretion system.

The process of DNA donation for natural transformation of bacteria is poorly understood and has been assumed to involve bacterial cell death. Recently in Neisseria gonorrhoeae we found that mutations in three genes in the gonococcal genetic island (GGI) reduced the ability of a strain to act as a donor in transformation and to release DNA into the culture. To better characterize the GGI and the process of DNA donation, the 57 kb genetic island was cloned, sequenced and subjected to insertional mutagenesis. DNA sequencing revealed that the GGI has characteristics of a horizontally acquired genomic island and encodes homologues of type IV secretion system proteins. The GGI was found to be incorporated near the chromosomal replication terminus at the dif site, a sequence targeted by the site-specific recombinase XerCD. Using a plasmid carrying a small region of the GGI and the associated dif site, we demonstrated that this model island could be integrated at the dif site in strains not carrying the GGI and was spontaneously excised from that site. Also, we were able to delete the entire 57 kb region by transformation with DNA from a strain lacking the GGI. Thus the GGI was likely acquired and integrated into the gonococcal chromosome by site-specific recombination and may be lost by site-specific recombination or natural transformation. We made mutations in six putative type IV secretion system genes and assayed these strains for the ability to secrete DNA. Five of the mutations greatly reduced or completely eliminated DNA secretion. Our data indicate that N. gonorrhoeae secretes DNA via a specific process. Donated DNA may be used in natural transformation, contributing to antigenic variation and the spread of antibiotic resistance, and it may modulate the host immune response.