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Random shuttle mutagenesis: gonococcal mutants deficient in pilin antigenic variation
Author(s) -
WIehr Ian J.,
Seifert H. Steven
Publication year - 1997
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1997.2971660.x
Subject(s) - biology , transposon mutagenesis , mutagenesis , genetics , transposable element , antigenic variation , insertional mutagenesis , pilin , shuttle vector , mutant , dna , sleeping beauty transposon system , microbiology and biotechnology , pilus , gene , recombinant dna , virulence , vector (molecular biology)
Summary Shuttle mutagenesis has been adapted to randomly mutate the genome of Neisseria gonorrhoeae (gono‐coccus; Gc). A size‐restricted plasmid library of Gc strain FA1090 was mutated with the mini‐transposon mTnEGNS. Randomness was tested by checking for transposon insertion bias between vector and insert DNA, Gc transformation efficiency of individual mutated clones, and representation of unique clones before and after Gc transformation with a mutated pool of DNA. Mutants created by random shuttle mutagenesis were screened, using a colony‐based polymerase chain 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‐associated phenotypes, including colony growth, natural DNA transformation competence, and repair of DNA damage caused by ultraviolet radiation.