Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation

Area of Science:

• Microbiology
• Genetics
• Molecular Biology

Background:

• Neisseria gonorrhoeae (gonococcus; Gc) possesses a complex genome that facilitates immune evasion through pilin antigenic variation.
• Understanding the genetic mechanisms underlying antigenic variation is crucial for developing effective treatments against gonorrhea.

Purpose of the Study:

• To adapt shuttle mutagenesis for random genome-wide mutation of Neisseria gonorrhoeae.
• To identify mutants deficient in pilin antigenic variation (Avd) and characterize their associated phenotypes.

Main Methods:

• A size-restricted plasmid library of Gc strain FA1090 was mutagenized using the mini-transposon mTnEGNS.
• Randomness of mutagenesis was assessed through various insertion bias and transformation efficiency tests.
• A colony-based PCR assay was employed to screen 8,064 mutants for defects in pilin antigenic variation.

Main Results:

• Twenty-two unique transposon insertion mutants exhibiting an antigenic variation deficient (Avd) phenotype were identified.
• These Avd mutants were classified into five types based on recombination defect-associated phenotypes.
• Phenotypes included alterations in colony growth, natural DNA transformation competence, and DNA repair following UV radiation damage.

Conclusions:

• Shuttle mutagenesis is an effective tool for generating random genomic mutations in Neisseria gonorrhoeae.
• The identified Avd mutants provide valuable genetic resources for studying the intricate mechanisms of pilin antigenic variation and DNA repair in gonococcus.