Use of rpsL as a Counterselectable Marker in Borrelia burgdorferi

Abstract

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.

FIG. 1.

Counterselection using rpsL. (A) Schematic of pBSrpsL. pBSrpsL was derived from pBSV2 by inserting the rpsL gene and its native promoter into the multiple cloning site. The plasmid also carries the kanamycin resistance open reading frame aphI under the control of the flgB promoter (flgBp) as well as an E. coli replication origin (ColE1) from pCR-XL-TOPO and a B. burgdorferi replication origin (cp9 rep) from the 9-kb circular plasmid cp9 (which includes genes bbc01, bbc02, and bbc03 plus two inverted repeats). (B) Model of counterselection. The DCSmR4 spirochetes (rounded rectangles) carry a mutant rpsL (encoding a K88E mutation in S12) on the chromosome that confers resistance to streptomycin (rpsL^R). The plasmid pBSrpsL carries the wild-type rpsL gene driven by its own promoter, which confers susceptibility to streptomycin (rpsL^S). B. burgdorferi DCSmR4 cells that have lost the pBSrpsL plasmid can be selected for with streptomycin.