Depletion of 32-kbp circular plasmids from Borrelia burgdorferi

The Lyme disease spirochete Borrelia burgdorferi has a very unusual genome composed of one linear chromosome and up to 21 linear and circular plasmids. Several plasmids are known to be important either for mammalian infection or tick colonization. A single spirochete harbors up to 7 different cp32 plasmids; however, nothing is known about their role in mammalian infection. The plasmids in this family are well maintained during in vitro cultivation, making it difficult to study their functions. To effectively deplete the plasmids, an 8kbp fragment containing essential elements for replication and partitioning in B. burgdorferi was amplified from one of the cp32 plasmids, cp32-3, and cloned into the vector pGE22 that carries a gentamycin resistance cassette and essential elements for replication in Escherichia coli. The resulting construct, pG22cp32-3plus, was electroporated into borrelial cells. By increasing gentamycin selection pressure, the spirochetes were forced to lose the corresponding cp32 plasmid. This strategy can be used to knock out other members of the cp32 family