Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi

Molecular genetic analysis of Borrelia burgdorferi, the cause of Lyme disea se, has been hampered by the absence of any means of efficient generation, identification, and complementation of chromosomal and plasmid null gene mu tants. The similarity of borrelial G + C content to that of Gram-positive o rganisms suggested that a wide-host-range plasmid active in Cram-positive b acteria might also be recognized by borrelial DNA replication machinery. On e such plasmid, pGK12, is able to propagate in both Gram-positive and Gram- negative bacteria and carries erythromycin and chloramphenicol resistance m arkers. pGK12 propagated extrachromosomally in B. burgdorferi B31 after ele ctroporation but conferred only erythromycin resistance. pGK12 was used to express enhanced green fluorescent protein in B31 under the control of the flaB promoter. Escherichia coli transformed with pGK12 DNA extracted from B 31 expressing only erythromycin resistance developed both erythromycin and chloramphenicol resistance, and plasmid DNA isolated from these transformed E. coli had a restriction pattern similar to the original pGK12, Our data indicate that the replicons of pGK12 can provide the basis to continue deve loping efficient genetic systems for B. burgdorferi together with the eryth romycin resistance and reporter egfp genes.