Efficient targeted mutagenesis in Borrelia burgdorferi

Genetic studies in Borrelia burgdorferi have been hindered by the lack of a nonborrelial selectable marker. Currently, the only selectable marker is g yrB(r), a mutated form of the chromosomal gyrB gene that encodes the B subu nit of DNA gyrase and confers resistance to the antibiotic coumermycin A(1) . The utility of the coumermycin-resistant gyrB(r) gene for targeted gene d isruption is limited by a high frequency of recombination with the endogeno us gyrB gene. A kanamycin resistance gene (kan) was introduced into B, burg dorferi, and its use as a selectable marker was explored in an effort to im prove the genetic manipulation of this pathogen. B. burgdorferi transforman ts 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 mere resistant to hig h levels of kanamycin. The kanamycin resistance marker allows efficient dir ect selection of mutants in B. burgdorferi and hence is a significant impro vement in the ability to construct isogenic mutant strains in this pathogen .