A double strand break does not promote Neisseria gonorrhoeae pilin antigenic variation

The major subunit of the Type IV pilus (T4p) of Neisseria gonorrhoeae undergoes Antigenic variation (Av) dependent on a guanine quadruplex (G4) DNA structure located upstream of the pilin gene. Since the presence of G4 DNA induces genome instability in both eukaryotic and prokaryotic chromosomes, we tested whether a double strand break (DSB) at the site of the pilE G4 sequence can substitute for G4 directed pilin Av. The G4 motif was replaced by an I-SceI cut site and the cut site was also introduced to locations near the origin of replication and the terminus. Expression of the I-SceI endonuclease from an irrelevant chromosomal site confirmed that the endonuclease functions to induce double strand breaks at all three locations. No antigenic variants were detected when the G4 was replaced with the I-SceI cut site, but there was a growth defect from having a DSB in the chromosome and suppressor mutations accumulated that were mainly deletions of the cut site and/or the entire pilE gene. Thus, the pilE G4 does not act to promote pilin Av by generating a DSB, but requires either a different type of break, nick, or more complex interactions with other factors to stimulate this programmed recombination system. Importance : Neisseria gonorrhoeae , the causative agent of gonorrhea, possesses a DNA recombination system to change one of its surface exposed antigens. This recombination system known as antigenic variation uses an alternate DNA structure to initiate variation. The guanine quadruplex DNA structure is known to cause nicks or breaks in DNA, however much remains unknown about how this structure functions in cells. We show that inducing a break by a different means does not allow for antigenic variation indicating that the DNA structure may have a more complicated role.