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 inactivati on, and the development of extrachromosomal cloning vectors, genetic analys is of Borrelia burgdorferi is becoming a reality. A previously described no nmotile, rod-shaped, kanamycin-resistant B. burgdorferi flaB::Km null mutan t was complemented by electroporation with the erythromycin resistance plas mid pED3 (a pGK12 derivative) containing the wild-type flaB sequence and 36 6 bp upstream from its initiation codon. The resulting MS17 clone possessed erythromycin and kanamycin resistance, flat-wave morphology, and microscop ic and macroscopic motility. Several other electroporations with plasmids c ontaining wild-type flaB and various lengths (198, 366, or 762 bp) of seque nce upstream from the flaB gene starting codon did not lead to functional r estoration of the nonmotile flaB null mutant. DNA hybridization, PCR analys is, and sequencing indicated that the wild-type flaB gene in nonmotile clon es was present in the introduced extrachromosomal plasmids, while the motil e 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 mechanism s for the failure of complementation for extrachromosomally located flaB ar e discussed.