TY - JOUR
T1 - Efficient targeted mutagenesis in Borrelia burgdorferi
AU - Bono, James L.
AU - Elias, Abdallah F.
AU - Kupko, John J.
AU - Stevenson, Brian
AU - Tilly, Kit
AU - Rosa, Patricia
PY - 2000/5
Y1 - 2000/5
N2 - Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumer-mycin A1. The utility of the coumer-mycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.
AB - Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is gyrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subunit of DNA gyrase and confers resistance to the antibiotic coumer-mycin A1. The utility of the coumer-mycin-resistant gyrB(r) gene for targeted gene disruption is limited by a high frequency of recombination with the endogenous gyrB gene. A kanamycin resistance gene (kan) was introduced into B. burgdorferi, and its use as a selectable marker was explored in an effort to improve the genetic manipulation of this pathogen. B. burgdorferi transformants with the kan gene expressed from its native promoter were susceptible to kanamycin. In striking contrast, transformants with the kan gene expressed from either the B. burgdorferi flaB or flgB promoter were resistant to high levels of kanamycin. The kanamycin resistance marker allows efficient direct selection of mutants in B. burgdorferi and hence is a significant improvement in the ability to construct isogenic mutant strains in this pathogen.
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U2 - 10.1128/JB.182.9.2445-2452.2000
DO - 10.1128/JB.182.9.2445-2452.2000
M3 - Article
C2 - 10762244
AN - SCOPUS:0034002143
SN - 0021-9193
VL - 182
SP - 2445
EP - 2452
JO - Journal of Bacteriology
JF - Journal of Bacteriology
IS - 9
ER -