title: Genetic transformation of the relapsing fever spirochete Borrelia hermsii: stable integration and expression of green fluorescent protein from linear plasmid 200.
abstract: Tick-borne relapsing fever (TBRF) is a spirochetal disease caused by at least 15 different Borrelia species. It is a serious human health concern in regions of endemicity throughout the world. Transmission to humans occurs through the bites of infected Ornithodoros ticks. In North America, the primary Borrelia species associated with human disease are B. hermsii and B. turicatae. Direct demonstration of the role of putative TBRF spirochete virulence factors in the disease process has been hindered by the lack of a genetic manipulation system and complete genome sequences. Expanding on recent developments in these areas, here we demonstrate the successful generation of a clone of B. hermsii YOR that constitutively produces green fluorescent protein (GFP) (B. hermsii YOR::kan gfp). This strain was generated through introduction of a kan-gfp cassette into a noncoding region of the 200-kb B. hermsii linear plasmid lp200. Genetic manipulation did not affect the growth rate or trigger the loss of native plasmids. B. hermsii YOR::kan gfp retained infectivity and elicited host seroconversion. Stable production of GFP was demonstrated both in vitro and in vivo. This study represents a significant step forward in the development of tools that can be employed to study the virulence mechanisms of TBRF spirochetes.
authors.affiliation: Department of Microbiology and Immunology, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA 23298-0678, USA.
authors.forename: Lindy M
authors.initials: LM
authors.lastname: Fine
authors.forename: Christopher G
authors.initials: CG
authors.lastname: Earnhart
authors.forename: Richard T
authors.initials: RT
authors.lastname: Marconi
mesh_terms: D000818:Animals; D000907:Antibodies, Bacterial / Q000097:blood; D001898:Borrelia / Q000235:genetics* / Q000254:growth & development / Q000276:immunology / Q000472:pathogenicity; D004195:Disease Models, Animal; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D042822:Genomic Instability; D049452:Green Fluorescent Proteins / Q000096:biosynthesis*; D051379:Mice; D008967:Molecular Biology / Q000379:methods*; D016254:Mutagenesis, Insertional; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D012061:Relapsing Fever / Q000382:microbiology; D014169:Transformation, Bacterial; D014774:Virulence
chemical_list: D000907:Antibodies, Bacterial; D049452:Green Fluorescent Proteins