Summary
The lack of a system for genetic manipulation has hindered studies on the molecular pathogenesis of relapsing fever Borrelia. The focus of this chapter is to describe selectable markers, manipulation strategies, and methods to electro-transform and clone wild-type infectious Borrelia hermsii. Preliminary studies suggest that the variable tick protein (Vtp) of B. hermsii is involved in tick-to-mammal transmission. To address this hypothesis, we have developed a system for genetic manipulation and have constructed clones of a Vtp mutant and an isogenic reconstituted strain. The methods described here are applicable for the inactivation of other loci in B. hermsii and should be adaptable for other species of relapsing fever spirochetes.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Falkow, S. (1988) Molecular Koch's postulates applied to microbial pathogenicity. Rev. Infect. Dis. 10 Suppl. 2, S274–276.
Dworkin, M.S., Schwan, T.G., and Anderson, D.E. (2002) Tick-borne relapsing fever in North America. Med. Clin. North Am. 86, 417–433.
Brouqui, P., Stein, A., Dupont, H.T., Gallian, P., Badiaga, S., Rolain, J.M., Mege, J.L., La Scola, B., Berbis, P., and Raoult, D. (2005) Ectoparasitism and vector-borne diseases in 930 homeless people from Marseilles. Medicine (Baltimore) 84, 61–68.
Cutler, S.J. (2006) Possibilities for relapsing fever reemergence. Emerg. Infect. Dis. 12, 369–374.
Carter, C.J., Bergström, S., Norris, S.J., and Barbour, A.G. (1994) A family of surface-exposed proteins of 20 kilodaltons in the genus Borrelia. Infect. Immun. 62, 2792–2799.
Schwan, T.G., and Hinnebusch, B.J. (1998) Bloodstream- versus tick-associated variants of a relapsing fever bacterium. Science 280, 1938–1940.
Schwan, T.G., Piesman, J., Golde, W.T., Dolan, M.C., and Rosa, P.A. (1995) Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc. Natl. Acad. Sci. USA 92, 2909–2913.
Grimm, D., Tilly, K., Byram, R., Stewart, P.E., Krum, J.G., Bueschel, D.M., Schwan, T.G., Policastro, P.F., Elias, A.F., and Rosa, P.A. (2004) Outer-surface protein C of the Lyme disease spirochete: a protein induced in ticks for infection of mammals. Proc. Natl. Acad. Sci. USA 101, 3142–3147.
Pal, U., Yang, X., Chen, M., Bockenstedt, L.K., Anderson, J.F., Flavell, R.A., Norgaard, M.V., and Fikrig, E. (2004) OspC facilitates Borrelia burgdorferi invasion of Ixodes scapularis salivary glands. J. Clin. Invest. 113, 220–230.
Tilly, K., Krum, J.G., Bestor, A., Jewett, M.W., Grimm, D., Bueschel, D., Byram, R., Dorward, D., Vanraden, M.J., Stewart, P., et al. (2006) Borrelia burgdorferi OspC protein required exclusively in a crucial early stage of mammalian infection. Infect. Immun. 74, 3554–3564.
Vieira, J., and Messing, J. (1991) New pUC-derived cloning vectors with different selectable markers and DNA replication origins. Gene 100, 189–194.
Stoenner, H.G., Dodd, T., and Larsen, C. (1982) Antigenic variation of Borrelia hermsii. J. Exp. Med. 156, 1297–1311.
Altschul, S.F., Gish, W., Miller, W., Myers, E.W., and Lipman, D.J. (1990) Basic local alignment search tool. J. Mol. Biol. 215, 403–410.
Stewart, P.E., Thalken, R., Bono, J.L., and Rosa, P. (2001) Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi. Mol. Microbiol. 39, 714–721.
Bono, J.L., Elias, A.F., Kupko, J.J., III, Stevenson, B., Tilly, K., and Rosa, P. (2000) Efficient targeted mutagenesis in Borrelia burgdorferi. J. Bacteriol. 182, 2445–2452.
Elias, A.F., Bono, J.L., Kupko, J.J., Stewart, P.E., Krum, J.G., and Rosa, P.A. (2003) New antibiotic resistance cassettes suitable for genetic studies in Borrelia burgdorferi. J. Mol. Microbiol. Biotechnol. 6, 29–40.
Fraser, C.M., Casjens, S., Huang, W.M., Sutton, G.G., Clayton, R., Lathigra, R., White, O., Ketchum, K.A., Dodson, R., Hickey, E.K., et al. (1997) Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580–586.
Casjens, S., Palmer, N., van Vugt, R., Huang, W.M., Stevenson, B., Rosa, P., Lathigra, R., Sutton, G., Peterson, J., Dodson, R.J., et al. (2000) A bacterial genome in flux: the twelve linear and nine circular extrachromosomal DNAs in an infectious isolate of the Lyme disease spirochete Borrelia burgdorferi. Mol. Microbiol. 35, 490–516.
Dai, Q., Restrepo, B.I., Porcella, S.F., Raffel, S.J., Schwan, T.G., and Barbour, A.G. (2006) Antigenic variation by Borrelia hermsii occurs through recombination between extragenic repetitive elements on linear plasmids. Mol. Microbiol. 60, 1329–1343.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specific enzymatic amplification of DNA in vitro: the polymerase chain reaction. Cold Spring Harb. Symp. Quant. Biol. 51, 263–273.
Samuels, D.S. (1995) Electrotransformation of the spirochete Borrelia burgdorferi, in Methods in Molecular Biology (Nickoloff, J.A., ed.), Humana, Totowa, NJ, pp. 253–259.
Tilly, K., Elias, A.F., Bono, J.L., Stewart, P., and Rosa, P. (2000) DNA exchange and insertional inactivation in spirochetes. J. Mol. Microbiol. Biotechnol. 2, 433–442.
Elias, A.F., Stewart, P.E., Grimm, D., Caimano, M.J., Eggers, C.H., Tilly, K., Bono, J.L., Akins, D.R., Radolf, J.D., Schwan, T.G., et al. (2002) Clonal polymorphism of Borrelia burgdorferi strain B31 MI: implications for mutagenesis in an infectious strain background. Infect. Immun. 70, 2139–2150.
Hinneb usch, B.J., Barbour, A.G., Restrepo, B.I., and Schwan, T.G. (1998) Population structure of the relapsing fever spirochete Borrelia hermsii as indicated by polymorphism of two multigene families that encode immunogenic outer surface lipoproteins. Infect. Immun. 66, 432–440.
Acknowledgments
We thank Merry Schrumpf, Kevin Lawrence, Aimee Giessler, and Gail Sylva for excellent technical assistance; Patti Rosa, Jim Bono, Abe Elias, and Philip Stewart for the generous donation of plasmid constructs and advice regarding genetic manipulation of Borrelia; Steve Porcella for assisting with B. hermsii sequences; and Mike Minnick for critical review of this text. The animal use protocol utilized in this study was approved by the Animal Care and Use Committee at the Rocky Mountain Laboratories. This work was supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2008 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Battisti, J.M., Raffel, S.J., Schwan, T.G. (2008). A System for Site-Specific Genetic Manipulation of the Relapsing Fever SpirocheteBorrelia hermsii . In: DeLeo, F.R., Otto, M. (eds) Bacterial Pathogenesis. Methods in Molecular Biology™, vol 431. Humana Press. https://doi.org/10.1007/978-1-60327-032-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-60327-032-8_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-740-2
Online ISBN: 978-1-60327-032-8
eBook Packages: Springer Protocols