1887

Abstract

We have used short oligonucleotides to genetically transform the Lyme disease spirochaete The oligonucleotides are derived from the sequence of an Arg-133 to He mutant (chromosomal) gene that confers resistance to the antibiotic coumermycin A. Oligonucleotides were about 10000-fold less efficient at transformation, on a molar basis, than longer PCR-generated substrates. All of the transformants tested contained the predicted site-directed silent mutation in their genes. Antisense oligonucleotides were more efficient at transformation than either sense or double-stranded oligonucleotides. This is the first demonstration of oligonucleotides used to introduce site-directed mutations directly into the genome of a bacterium.

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/content/journal/micro/10.1099/00221287-143-2-519
1997-02-01
2024-04-19
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References

  1. Baranton G., Old I.G. 1995; The Spirochaetes: a different way of life.. Bull Inst Pasteur 93:63–95
    [Google Scholar]
  2. Barbour A.G., Fish D. 1993; The biological and social phenomenon of Lyme disease.. Science 260:1610–1616
    [Google Scholar]
  3. Hinnebusch J., Tilly K. 1993; Linear plasmids and chromosomes in bacteria.. Mol Microbiol 10:917–922
    [Google Scholar]
  4. Maxwell A. 1993; The interaction between coumarin drugs and DNA gyrase.. Mol Microbiol 9:681–686
    [Google Scholar]
  5. Moerschell R.P., Tsunasawa S., Sherman F. 1988; Transformation of yeast with synthetic oligonucleotides.. Proc Natl Acad Sci USA 85524–528
    [Google Scholar]
  6. Nickoloff J.A. editor 1995; Electroporation protocols for microorganisms.. In Methods in Molecular Biology p. 372 Walker J.M. Series Edited by Totowa, New Jersey: Humana Press;
    [Google Scholar]
  7. Rosa P.A., Hogan D.M. 1992; Colony formation by Borrelia burgdorferi in solid medium: clonal analysis of osp locus variants.. In First International Conference on Tick-Borne Pathogens at the Host-Vector Interface: An Agenda for Research pp. 95–103 Munderloh U.G., Kurtti T.J. Edited by St Paul: University of Minnesota;
    [Google Scholar]
  8. Rosa P., Samuels D.S., Hogan D., Stevenson B., Casjens S., Tilly K. 1996; Directed insertion of a selectable marker into a circular plasmid of Borrelia burgdorferi. . J Bacteriol 178:5946–5953
    [Google Scholar]
  9. Saint Girons I., Old I.G., Davidson B.E. 1994; Molecular biology of the Borrelia, bacteria with linear replicons.. Microbiology 140:1803–1816
    [Google Scholar]
  10. Samuels D.S. 1995; Electrotransformation of the spirochete Borrelia burgdorferi. . In Electroporation Protocols for Micro-organisms pp. 253–259 Nickoloff J.A. Edited by Totowa, New Jersey: Humana Press;
    [Google Scholar]
  11. Samuels D.S., Garon C.F. 1993; Coumermycin A1 inhibits growth and induces relaxation of supercoiled plasmids in Borrelia burgdorferi, the Lyme disease agent.. Antimicrob Agents Chemother 37:46–50
    [Google Scholar]
  12. Samuels D.S., Mach K.E., Garon C.F. 1994a; Genetic transformation of the Lyme disease agent Borrelia burgdorferi with coumarin-resistant gyrB. . J Bacteriol 176:6045–6049
    [Google Scholar]
  13. Samuels D.S., Marconi R.T., Huang W.M., Garon C.F. 1994b; gyrB mutations in coumermycin A1-resistant Borrelia burgdorferi. . J Bacteriol 176:3072–3075
    [Google Scholar]
  14. Shigekawa K., Dower W.J. 1988; Electroporation of eukaryotes and prokaryotes: a general approach to the introduction of macromolecules into cells.. BioTechniques 6:742–751
    [Google Scholar]
  15. Steere A.C. 1994; Lyme disease: a growing threat to urban populations.. Proc Natl Acad Sci USA 912378–2383
    [Google Scholar]
  16. Trevors J.T., Chassy B.M., Dower W.J., Blaschek H.P. 1992; Electrotransformation of bacteria by plasmid DNA.. In Guide to Electroporation and Electrofusion pp. 265–290 Chang D.C., Chassy B.M., Saunders J.A., Sowers A.E. Edited by San Diego: Academic Press;
    [Google Scholar]
  17. Yamamoto T., Moerschell R.P., Wakem L.P., Komar-Panicucci S., Sherman F. 1992; Strand-specificity in the transformation of yeast with synthetic oligonucleotides.. Genetics 131:811–819
    [Google Scholar]
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