Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi.
Borrelia burgdorferi contains abundant circular and linear plasmids, but the mechanism of replication of these extrachromosomal elements is unknown. A B. burgdorferi 9 kb circular plasmid (cp9) was amplified in its entirety by the polymerase chain reaction and used to construct a shuttle vector that replicates in Escherichia coli and B. burgdorferi. A 3.3 kb region of cp9 containing three open reading frames was used to construct a smaller shuttle vector, designated pBSV2. This vector was stably maintained in B. burgdorferi, indicating that all elements necessary for autonomous replication are probably located on this 3.3 kb fragment. A non-infectious B. burgdorferi strain was efficiently transformed by pBSV2. Additionally, infectious B. burgdorferi was also successfully transformed by pBSV2, indicating that infectious strains of this important human pathogen can now be genetically manipulated.
Stewart
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Laboratory of Human Bacterial Pathogenesis, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840, USA. pestewart@niaid.nih.gov
Thalken
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Bono
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Rosa
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D000818:Animals; D015139:Blotting, Southern; D015748:Borrelia burgdorferi Group / Q000235:genetics* / Q000254:growth & development / Q000472:pathogenicity; D004261:DNA Replication; D004269:DNA, Bacterial / Q000235:genetics; D004270:DNA, Circular / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D006801:Humans; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014169:Transformation, Bacterial
D004269:DNA, Bacterial; D004270:DNA, Circular