RANDOM SHUTTLE MUTAGENESIS - GONOCOCCAL MUTANTS DEFICIENT IN PILIN ANTIGENIC VARIATION

Shuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gonococcus; Gc). A size-restricted plasmid libr ary of Gc strain FA1090 was mutated with the mini-transposon mTnEGNS. Randomness was tested by checking for transposon insertion bias betwee n vector and insert DNA, Gc transformation efficiency of individual mu tated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random s huttle mutagenesis were screened, using a colony-based polymerase chai n reaction assay, for the ability to undergo pilin antigenic variation . Out of 8064 mutants screened, 22 unique transposon insertion mutants were found to be antigenic variation deficient (Avd). The Avd mutants were separated into five types according to recombination defect-asso ciated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.