Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Jun;178(11):3293–3307. doi: 10.1128/jb.178.11.3293-3307.1996

Borrelia burgdorferi supercoiled plasmids encode multicopy tandem open reading frames and a lipoprotein gene family.

S F Porcella 1, T G Popova 1, D R Akins 1, M Li 1, J D Radolf 1, M V Norgard 1
PMCID: PMC178083  PMID: 8655511

Abstract

DNA sequencing and Southern blot analyses of a Borrelia burgdorferi DNA fragment encoding a signal sequence led to the discovery of a genetic locus, designated 2.9, which appears to be present in at least seven copies in virulent B. burgdorferi 297. DNA sequence analysis of these regions revealed that each 2.9 locus contained an operon of four genes (ABCD) and open reading frames designated rep+ (positive strand) and rep- (negative strand) which encoded multiple repeat motifs. Downstream of the rep+ gene(s) in six of the completely cloned and sequenced 2.9 loci also were lipoprotein (LP) genes possessing highly similar signal sequences but encoding variable mature polypeptides. The lipoproteins could he separated into two classes on the basis of hydrophilicity profiles, sequence similarities, and reactivity with specific antibodies. The 2.9 loci were localized to two (20- and 30-kb) supercoiled plasmids in B. burgdorferi 297. Northern (RNA) blot analysis established that the 2.9 ABCD operon was only minimally expressed, whereas the rep- gene(s) and at least three of the seven LP genes were expressed by B. burgdorferi in vitro. A single putative promoter element was identified by RNA primer extension analysis upstream of the ABCD operon, whereas a number of potential promoter regions existed upstream of the LP genes. The combined data indicate that the ABCD operon, rep+ and rep- genes, and LP genes are separately transcribed during in vitro growth. The 2.9 loci possess a repetitiveness, diversity, and complexity not previously described for B. burgdorferi; differential expression of these genes may facilitate the spirochete's ability to survive in diverse host environments.

Full Text

The Full Text of this article is available as a PDF (1.0 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akins D. R., Porcella S. F., Popova T. G., Shevchenko D., Baker S. I., Li M., Norgard M. V., Radolf J. D. Evidence for in vivo but not in vitro expression of a Borrelia burgdorferi outer surface protein F (OspF) homologue. Mol Microbiol. 1995 Nov;18(3):507–520. doi: 10.1111/j.1365-2958.1995.mmi_18030507.x. [DOI] [PubMed] [Google Scholar]
  2. Aron L., Alekshun M., Perlee L., Schwartz I., Godfrey H. P., Cabello F. C. Cloning and DNA sequence analysis of bmpC, a gene encoding a potential membrane lipoprotein of Borrelia burgdorferi. FEMS Microbiol Lett. 1994 Oct 15;123(1-2):75–82. doi: 10.1111/j.1574-6968.1994.tb07204.x. [DOI] [PubMed] [Google Scholar]
  3. Barbour A. G. Antigenic variation of a relapsing fever Borrelia species. Annu Rev Microbiol. 1990;44:155–171. doi: 10.1146/annurev.mi.44.100190.001103. [DOI] [PubMed] [Google Scholar]
  4. Barbour A. G. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med. 1984 Jul-Aug;57(4):521–525. [PMC free article] [PubMed] [Google Scholar]
  5. Barbour A. G. Plasmid analysis of Borrelia burgdorferi, the Lyme disease agent. J Clin Microbiol. 1988 Mar;26(3):475–478. doi: 10.1128/jcm.26.3.475-478.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bergström S., Bundoc V. G., Barbour A. G. Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi. Mol Microbiol. 1989 Apr;3(4):479–486. doi: 10.1111/j.1365-2958.1989.tb00194.x. [DOI] [PubMed] [Google Scholar]
  7. Brandt M. E., Riley B. S., Radolf J. D., Norgard M. V. Immunogenic integral membrane proteins of Borrelia burgdorferi are lipoproteins. Infect Immun. 1990 Apr;58(4):983–991. doi: 10.1128/iai.58.4.983-991.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bundoc V. G., Barbour A. G. Clonal polymorphisms of outer membrane protein OspB of Borrelia burgdorferi. Infect Immun. 1989 Sep;57(9):2733–2741. doi: 10.1128/iai.57.9.2733-2741.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Champion C. I., Blanco D. R., Skare J. T., Haake D. A., Giladi M., Foley D., Miller J. N., Lovett M. A. A 9.0-kilobase-pair circular plasmid of Borrelia burgdorferi encodes an exported protein: evidence for expression only during infection. Infect Immun. 1994 Jul;62(7):2653–2661. doi: 10.1128/iai.62.7.2653-2661.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunn J. J., Buchstein S. R., Butler L. L., Fisenne S., Polin D. S., Lade B. N., Luft B. J. Complete nucleotide sequence of a circular plasmid from the Lyme disease spirochete, Borrelia burgdorferi. J Bacteriol. 1994 May;176(9):2706–2717. doi: 10.1128/jb.176.9.2706-2717.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Engstrom S. M., Shoop E., Johnson R. C. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. J Clin Microbiol. 1995 Feb;33(2):419–427. doi: 10.1128/jcm.33.2.419-427.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ferdows M. S., Barbour A. G. Megabase-sized linear DNA in the bacterium Borrelia burgdorferi, the Lyme disease agent. Proc Natl Acad Sci U S A. 1989 Aug;86(15):5969–5973. doi: 10.1073/pnas.86.15.5969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Fuchs R., Jauris S., Lottspeich F., Preac-Mursic V., Wilske B., Soutschek E. Molecular analysis and expression of a Borrelia burgdorferi gene encoding a 22 kDa protein (pC) in Escherichia coli. Mol Microbiol. 1992 Feb;6(4):503–509. doi: 10.1111/j.1365-2958.1992.tb01495.x. [DOI] [PubMed] [Google Scholar]
  15. Garcia-Monco J. C., Benach J. L. Lyme neuroborreliosis. Ann Neurol. 1995 Jun;37(6):691–702. doi: 10.1002/ana.410370602. [DOI] [PubMed] [Google Scholar]
  16. Gassmann G. S., Kramer M., Göbel U. B., Wallich R. Nucleotide sequence of a gene encoding the Borrelia burgdorferi flagellin. Nucleic Acids Res. 1989 May 11;17(9):3590–3590. doi: 10.1093/nar/17.9.3590. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hechemy K. E., Samsonoff W. A., Harris H. L., McKee M. Adherence and entry of Borrelia burgdorferi in Vero cells. J Med Microbiol. 1992 Apr;36(4):229–238. doi: 10.1099/00222615-36-4-229. [DOI] [PubMed] [Google Scholar]
  18. Hoffman C. S., Wright A. Fusions of secreted proteins to alkaline phosphatase: an approach for studying protein secretion. Proc Natl Acad Sci U S A. 1985 Aug;82(15):5107–5111. doi: 10.1073/pnas.82.15.5107. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Jonsson M., Bergström S. Transcriptional and translational regulation of the expression of the major outer surface proteins in Lyme disease Borrelia strains. Microbiology. 1995 Jun;141(Pt 6):1321–1329. doi: 10.1099/13500872-141-6-1321. [DOI] [PubMed] [Google Scholar]
  20. Kenney T. J., York K., Youngman P., Moran C. P., Jr Genetic evidence that RNA polymerase associated with sigma A factor uses a sporulation-specific promoter in Bacillus subtilis. Proc Natl Acad Sci U S A. 1989 Dec;86(23):9109–9113. doi: 10.1073/pnas.86.23.9109. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kurtti T. J., Munderloh U. G., Krueger D. E., Johnson R. C., Schwan T. G. Adhesion to and invasion of cultured tick (Acarina: Ixodidae) cells by Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) and maintenance of infectivity. J Med Entomol. 1993 May;30(3):586–596. doi: 10.1093/jmedent/30.3.586. [DOI] [PubMed] [Google Scholar]
  22. Lam T. T., Nguyen T. P., Montgomery R. R., Kantor F. S., Fikrig E., Flavell R. A. Outer surface proteins E and F of Borrelia burgdorferi, the agent of Lyme disease. Infect Immun. 1994 Jan;62(1):290–298. doi: 10.1128/iai.62.1.290-298.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Lane R. S., Piesman J., Burgdorfer W. Lyme borreliosis: relation of its causative agent to its vectors and hosts in North America and Europe. Annu Rev Entomol. 1991;36:587–609. doi: 10.1146/annurev.en.36.010191.003103. [DOI] [PubMed] [Google Scholar]
  24. Liveris D., Gazumyan A., Schwartz I. Molecular typing of Borrelia burgdorferi sensu lato by PCR-restriction fragment length polymorphism analysis. J Clin Microbiol. 1995 Mar;33(3):589–595. doi: 10.1128/jcm.33.3.589-595.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Logigian E. L., Kaplan R. F., Steere A. C. Chronic neurologic manifestations of Lyme disease. N Engl J Med. 1990 Nov 22;323(21):1438–1444. doi: 10.1056/NEJM199011223232102. [DOI] [PubMed] [Google Scholar]
  26. Margolis N., Hogan D., Tilly K., Rosa P. A. Plasmid location of Borrelia purine biosynthesis gene homologs. J Bacteriol. 1994 Nov;176(21):6427–6432. doi: 10.1128/jb.176.21.6427-6432.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Moody K. D., Barthold S. W., Terwilliger G. A. Lyme borreliosis in laboratory animals: effect of host species and in vitro passage of Borrelia burgdorferi. Am J Trop Med Hyg. 1990 Jul;43(1):87–92. doi: 10.4269/ajtmh.1990.43.87. [DOI] [PubMed] [Google Scholar]
  28. Mulligan M. E., Hawley D. K., Entriken R., McClure W. R. Escherichia coli promoter sequences predict in vitro RNA polymerase selectivity. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 2):789–800. doi: 10.1093/nar/12.1part2.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Munderloh U. G., Park Y. J., Dioh J. M., Fallon A. M., Kurtti T. J. Plasmid modifications in a tick-borne pathogen, Borrelia burgdorferi, cocultured with tick cells. Insect Mol Biol. 1993;1(4):195–203. doi: 10.1111/j.1365-2583.1993.tb00092.x. [DOI] [PubMed] [Google Scholar]
  30. Norris S. J., Carter C. J., Howell J. K., Barbour A. G. Low-passage-associated proteins of Borrelia burgdorferi B31: characterization and molecular cloning of OspD, a surface-exposed, plasmid-encoded lipoprotein. Infect Immun. 1992 Nov;60(11):4662–4672. doi: 10.1128/iai.60.11.4662-4672.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Ojaimi C., Davidson B. E., Saint Girons I., Old I. G. Conservation of gene arrangement and an unusual organization of rRNA genes in the linear chromosomes of the Lyme disease spirochaetes Borrelia burgdorferi, B. garinii and B. afzelii. Microbiology. 1994 Nov;140(Pt 11):2931–2940. doi: 10.1099/13500872-140-11-2931. [DOI] [PubMed] [Google Scholar]
  32. Pugsley A. P., Kornacker M. G., Poquet I. The general protein-export pathway is directly required for extracellular pullulanase secretion in Escherichia coli K12. Mol Microbiol. 1991 Feb;5(2):343–352. doi: 10.1111/j.1365-2958.1991.tb02115.x. [DOI] [PubMed] [Google Scholar]
  33. Pugsley A. P. The complete general secretory pathway in gram-negative bacteria. Microbiol Rev. 1993 Mar;57(1):50–108. doi: 10.1128/mr.57.1.50-108.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ray C., Igo M., Shafer W., Losick R., Moran C. P., Jr Suppression of ctc promoter mutations in Bacillus subtilis. J Bacteriol. 1988 Feb;170(2):900–907. doi: 10.1128/jb.170.2.900-907.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Reindl M., Redl B., Stöffler G. Isolation and analysis of a linear plasmid-located gene of Borrelia burgdorferi B29 encoding a 27 kDa surface lipoprotein (P27) and its overexpression in Escherichia coli. Mol Microbiol. 1993 Jun;8(6):1115–1124. doi: 10.1111/j.1365-2958.1993.tb01656.x. [DOI] [PubMed] [Google Scholar]
  36. Sadziene A., Wilske B., Ferdows M. S., Barbour A. G. The cryptic ospC gene of Borrelia burgdorferi B31 is located on a circular plasmid. Infect Immun. 1993 May;61(5):2192–2195. doi: 10.1128/iai.61.5.2192-2195.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Saint Girons I., Old I. G., Davidson B. E. Molecular biology of the Borrelia, bacteria with linear replicons. Microbiology. 1994 Aug;140(Pt 8):1803–1816. doi: 10.1099/13500872-140-8-1803. [DOI] [PubMed] [Google Scholar]
  38. Schwan T. G., Burgdorfer W., Garon C. F. Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect Immun. 1988 Aug;56(8):1831–1836. doi: 10.1128/iai.56.8.1831-1836.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Schwan T. G., Piesman J., Golde W. T., Dolan M. C., Rosa P. A. Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2909–2913. doi: 10.1073/pnas.92.7.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Shoberg R. J., Thomas D. D. Specific adherence of Borrelia burgdorferi extracellular vesicles to human endothelial cells in culture. Infect Immun. 1993 Sep;61(9):3892–3900. doi: 10.1128/iai.61.9.3892-3900.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Simpson W. J., Cieplak W., Schrumpf M. E., Barbour A. G., Schwan T. G. Nucleotide sequence and analysis of the gene in Borrelia burgdorferi encoding the immunogenic P39 antigen. FEMS Microbiol Lett. 1994 Jun 15;119(3):381–387. doi: 10.1111/j.1574-6968.1994.tb06917.x. [DOI] [PubMed] [Google Scholar]
  42. Simpson W. J., Garon C. F., Schwan T. G. Borrelia burgdorferi contains repeated DNA sequences that are species specific and plasmid associated. Infect Immun. 1990 Apr;58(4):847–853. doi: 10.1128/iai.58.4.847-853.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sinsky R. J., Piesman J. Ear punch biopsy method for detection and isolation of Borrelia burgdorferi from rodents. J Clin Microbiol. 1989 Aug;27(8):1723–1727. doi: 10.1128/jcm.27.8.1723-1727.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Steere A. C. Lyme disease. N Engl J Med. 1989 Aug 31;321(9):586–596. doi: 10.1056/NEJM198908313210906. [DOI] [PubMed] [Google Scholar]
  45. Suk K., Das S., Sun W., Jwang B., Barthold S. W., Flavell R. A., Fikrig E. Borrelia burgdorferi genes selectively expressed in the infected host. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4269–4273. doi: 10.1073/pnas.92.10.4269. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Swancutt M. A., Radolf J. D., Norgard M. V. The 34-kilodalton membrane immunogen of Treponema pallidum is a lipoprotein. Infect Immun. 1990 Feb;58(2):384–392. doi: 10.1128/iai.58.2.384-392.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Swancutt M. A., Riley B. S., Radolf J. D., Norgard M. V. Molecular characterization of the pathogen-specific, 34-kilodalton membrane immunogen of Treponema pallidum. Infect Immun. 1989 Nov;57(11):3314–3323. doi: 10.1128/iai.57.11.3314-3323.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Wallich R., Brenner C., Kramer M. D., Simon M. M. Molecular cloning and immunological characterization of a novel linear-plasmid-encoded gene, pG, of Borrelia burgdorferi expressed only in vivo. Infect Immun. 1995 Sep;63(9):3327–3335. doi: 10.1128/iai.63.9.3327-3335.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zückert W. R., Meyer J. Circular and linear plasmids of Lyme disease spirochetes have extensive homology: characterization of a repeated DNA element. J Bacteriol. 1996 Apr;178(8):2287–2298. doi: 10.1128/jb.178.8.2287-2298.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES