We have demonstrated that rpsL, encoding the S12 protein of the small ribosomal subunit, can be used as a counterselectable marker in Borrelia burgdorferi, the causative agent of
Lyme disease. Mutations in rpsL confer streptomycin resistance. Streptomycin susceptibility is dominant in an rpsL merodiploid, and streptomycin selects for the loss of wild-type rpsL carried in trans. This is the first description of a counterselectable marker in B. burgdorferi.