Complementation of a nonmotile flaB mutant of Borrelia burgdorferi by chromosomal integration of a plasmid containing a wild-type flaB allele.
With the recent identification of antibiotic resistance phenotypes, the use of reporter genes, the isolation of null mutants by insertional inactivation, and the development of extrachromosomal cloning vectors, genetic analysis of Borrelia burgdorferi is becoming a reality. A previously described nonmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutant was complemented by electroporation with the erythromycin resistance plasmid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 366 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscopic and macroscopic motility. Several other electroporations with plasmids containing wild-type flaB and various lengths (198, 366, or 762 bp) of sequence upstream from the flaB gene starting codon did not lead to functional restoration of the nonmotile flaB null mutant. DNA hybridization, PCR analysis, and sequencing indicated that the wild-type flaB gene in nonmotile clones was present in the introduced extrachromosomal plasmids, while the motile MS17 clone was a merodiploid containing single tandem chromosomal copies of mutated flaB::Km and wild-type flaB with a 366-bp sequence upstream from its starting codon. Complementation was thus achieved only when wild-type flaB was inserted into the borrelial chromosome. Several possible mechanisms for the failure of complementation for extrachromosomally located flaB are discussed.
Sartakova
M L
ML
Department of Microbiology, New York Medical College, Valhalla, New York 10595, USA.
Dobrikova
E Y
EY
Motaleb
M A
MA
Godfrey
H P
HP
Charon
N W
NW
Cabello
F C
FC
D000483:Alleles; D025065:Borrelia burgdorferi / Q000235:genetics* / Q000378:metabolism; D002876:Chromosomes, Bacterial / Q000235:genetics*; D018274:Electroporation; D005408:Flagellin / Q000235:genetics*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D014169:Transformation, Bacterial
D005408:Flagellin; C416532:flaB flagellin