BBE02 Disruption Mutants of Borrelia burgdorferi B31 Have a Highly Transformable, Infectious Phenotype

Abstract

We constructed highly transformable and infectious Borrelia burgdorferi B31 by inactivating BBE02, a putative restriction-modification gene on the linear plasmid lp25. The low-passage-number B31 clones 5A4 (containing all plasmids) and 5A18 (lp28-4 ⁻ lp56 ⁻ ) were used for this study, and BBE02 was disrupted by homologous recombination. The transformation efficiency with the shuttle vector pBSV2 C03 :: gntΔkan was increased from r and from 14 to approximately 600 colonies per μg of DNA for 5A18 and 5A18 BBE02::Kan ^r . lp25, which is required for infectivity in mice, was retained in BBE02 mutants transformed with pBSV2 C03 :: gntΔkan , but lp25 was not detected in transformants of the parental clones 5A4 and 5A18. BBE02 disruptants and pBSV2 C03 :: gntΔkan transformants of these clones remained infectious in C3H/HeN mice, and the 50% infective doses of the BBE02 disruptants were 2 organisms per mouse. The inactivation of BBE02 thus eliminates a transformation barrier for infectious B. burgdorferi B31 and will provide a valuable tool for studying the virulence factors of Lyme disease.