DIRECTED INSERTION OF A SELECTABLE MARKER INTO A CIRCULAR PLASMID OF BORRELIA-BURGDORFERI

Studies of the biology of Borrelia burgdorferi and the pathogenesis of Lyme disease are severely limited by the current lack of genetic tool s. As an initial step toward facile genetic manipulation of this patho genic spirochete, we have investigated gene inactivation by allelic ex change using a mutated borrelial gyrB gene that confers resistance to the antibiotic coumermycin A(1) as a selectable marker. We have transf ormed B. burgdorferi by electroporation with a linear fragment of DNA in which this selectable marker was flanked by sequences from a native borrelial 26-kb circular plasmid. We have identified coumermycin A(1) -resistant transformants in which gyrB had interrupted the targeted si te on the 26-kb plasmid via homologous recombination with the flanking sequences. Antibiotic resistance conferred by the mutated gyrB gene o n the plasmid is dominant, and transformed spirochetes carrying this p lasmid do not contain any unaltered copies of the plasmid. Coumermycin A(1) resistance can be transferred to naive B. burgdorferi by transfo rmation with borrelial plasmid DNA from the initial transformants. Thi s work represents the first example of a directed mutation in B. burgd orferi whereby a large segment of heterologous DNA (gyrB) has been ins erted via homologous recombination with flanking sequences, thus demon strating the feasibility of specific gene inactivation by allelic exch ange.