A DNA REPAIR/PHASE VARIATION REPORTER SYSTEM USING A POLY-GUANINE TRACT IN A NEISSERIA GONORRHOEAE NITROREDUCTASE GENE

Abstract

Neisseria gonorrhoeae undergoes phase variation to adapt to new environments, increase pathogenesis, and evade the host immune system. This may be due to defects in DNA repair. A reporter system was created to detect phase variation by phenotypic switching from a nitrofuran-sensitive phenotype to a nitrofuran-resistant (NitR) phenotype. Strains were created with poly-guanine tracts from 5 to 12 guanines in the coding region of a nitroreductase gene (nfsB) that would be susceptible to frame-shifting mutations during DNA replication. The minimum number of consecutive guanines needed to observe increased mutation was 5. A strain expressing 7 guanines nfsB possessed nitroreductase activity similar to wild-type and a spontaneous mutation frequency that was increased ~104 fold relative to wild-type. Frame-shifting mutations of strain expressing 8 guanines in nfsB were observed using denaturing gradient gel electrophoresis (DGGE). Future work with the reporter system could lead to new understanding of phase variation and DNA repair.