Restriction–modification systems are required for Neisseria gonorrhoeae pilin antigenic variation

(Gc) pilin antigenic variation is a diversity-generating system that uses gene conversion to produce numerous PilE protein variants, the major subunit of the Type IV pilus (T4p). Pilin antigenic variation allows the bacteria to escape immune surveillance by expressing a variant but functional pilus, and can also alter T4p expression. While pilin antigenic variation requires many conserved homologous recombination and DNA repair factors, the pattern of sequence changes leading to pilin antigenic variants resembles that of an annealing reaction, rather than the expected long recombination tracts usually found in homologous recombination. We demonstrate that two paralogous restriction-modification modules cleave specific, unmodified sequences within the expressed and silent pilin loci and that cleavage is an important process for pilin antigenic variation. Moreover, expression of these restriction activities in a subset of the bacterial population affects the fitness. These findings partially explain the patchwork recombination patterns of pilin antigenic variants and show a unique mechanism for generating diversity.