Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Apr;179(7):2289–2299. doi: 10.1128/jb.179.7.2289-2299.1997

Molecular characterization of a large Borrelia burgdorferi motility operon which is initiated by a consensus sigma70 promoter.

Y Ge 1, I G Old 1, I Saint Girons 1, N W Charon 1
PMCID: PMC178966  PMID: 9079915

Abstract

A large motility operon, referred to as the flgB operon, was identified, characterized, and mapped at 310 to 320 kb on the linear chromosome of the spirochete Borrelia burgdorferi. This is the first report that a sigma70-like promoter rather than a sigma28-like promoter is involved in the transcription of a major motility operon in bacteria. From these results in conjunction with results from a previous study (Y. Ge and N. W. Charon, Gene, in press), we have identified 26 genes in this operon that are relevant to motility and flagellar synthesis. With few exceptions, the gene order and deduced gene products were most similar to those of other spirochetes and Bacillus subtilis. Primer extension analysis indicated that transcription initiated from a conserved sigma70-like promoter immediately upstream of flgB; this promoter mapped within the heat-shock-induced protease gene hslU. Reverse transcriptase PCR analysis indicated that a single transcript of 21 kb initiated at this promoter and extended through flgE and (with our previous results) onto the putative motility gene flbE. The flgB promoter element had strong activity in both Escherichia coli and Salmonella typhimurium. As expected, a mutant of S. typhimurium with an inactivated flagellum-specific sigma28 factor did not affect the function of this promoter. Western blot analysis indicated that B. burgdorferi recombinant FliG and FliI were antigenically similar to those of E. coli and other spirochetes. Although complementation of E. coli or S. typhimurium fliG or fliI mutants with the B. burgdorferi genes was unsuccessful, B. burgdorferi recombinant FliI completely inhibited flagellar synthesis and motility of wild-type E. coli and S. typhimurium. These results show that spirochete motility genes can influence flagellar synthesis in other species of bacteria. Finally, Western blot analysis with sera from infected humans and animals indicated a weak or nondetectable response to recombinant FliG and FliI. These results indicate that these antigens are not favorable candidate reagents to be used in the diagnosis of Lyme disease.

Full Text

The Full Text of this article is available as a PDF (1.1 MB).

Selected References

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

  1. Aizawa S. I. Flagellar assembly in Salmonella typhimurium. Mol Microbiol. 1996 Jan;19(1):1–5. doi: 10.1046/j.1365-2958.1996.344874.x. [DOI] [PubMed] [Google Scholar]
  2. Akerley B. J., Cotter P. A., Miller J. F. Ectopic expression of the flagellar regulon alters development of the Bordetella-host interaction. Cell. 1995 Feb 24;80(4):611–620. doi: 10.1016/0092-8674(95)90515-4. [DOI] [PubMed] [Google Scholar]
  3. Albertini A. M., Caramori T., Crabb W. D., Scoffone F., Galizzi A. The flaA locus of Bacillus subtilis is part of a large operon coding for flagellar structures, motility functions, and an ATPase-like polypeptide. J Bacteriol. 1991 Jun;173(11):3573–3579. doi: 10.1128/jb.173.11.3573-3579.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Anderson D. K., Ohta N., Wu J., Newton A. Regulation of the Caulobacter crescentus rpoN gene and function of the purified sigma 54 in flagellar gene transcription. Mol Gen Genet. 1995 Mar 20;246(6):697–706. doi: 10.1007/BF00290715. [DOI] [PubMed] [Google Scholar]
  5. Assous M. V., Postic D., Paul G., Névot P., Baranton G. Western blot analysis of sera from Lyme borreliosis patients according to the genomic species of the Borrelia strains used as antigens. Eur J Clin Microbiol Infect Dis. 1993 Apr;12(4):261–268. doi: 10.1007/BF01967256. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Bischoff D. S., Ordal G. W. Bacillus subtilis chemotaxis: a deviation from the Escherichia coli paradigm. Mol Microbiol. 1992 Jan;6(1):23–28. doi: 10.1111/j.1365-2958.1992.tb00833.x. [DOI] [PubMed] [Google Scholar]
  8. Brosius J. Plasmid vectors for the selection of promoters. Gene. 1984 Feb;27(2):151–160. doi: 10.1016/0378-1119(84)90136-7. [DOI] [PubMed] [Google Scholar]
  9. Carpenter P. B., Zuberi A. R., Ordal G. W. Bacillus subtilis flagellar proteins FliP, FliQ, FliR and FlhB are related to Shigella flexneri virulence factors. Gene. 1993 Dec 31;137(2):243–245. doi: 10.1016/0378-1119(93)90014-t. [DOI] [PubMed] [Google Scholar]
  10. Casjens S., Delange M., Ley H. L., 3rd, Rosa P., Huang W. M. Linear chromosomes of Lyme disease agent spirochetes: genetic diversity and conservation of gene order. J Bacteriol. 1995 May;177(10):2769–2780. doi: 10.1128/jb.177.10.2769-2780.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Champion C. I., Miller J. N., Lovett M. A., Blanco D. R. Cloning, sequencing, and expression of two class B endoflagellar genes of Treponema pallidum subsp. pallidum encoding the 34.5- and 31.0-kilodalton proteins. Infect Immun. 1990 Jun;58(6):1697–1704. doi: 10.1128/iai.58.6.1697-1704.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Charon N. W., Goldstein S. F., Curci K., Limberger R. J. The bent-end morphology of Treponema phagedenis is associated with short, left-handed, periplasmic flagella. J Bacteriol. 1991 Aug;173(15):4820–4826. doi: 10.1128/jb.173.15.4820-4826.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Charon N. W., Greenberg E. P., Koopman M. B., Limberger R. J. Spirochete chemotaxis, motility, and the structure of the spirochetal periplasmic flagella. Res Microbiol. 1992 Jul-Aug;143(6):597–603. doi: 10.1016/0923-2508(92)90117-7. [DOI] [PubMed] [Google Scholar]
  14. Chen L., Helmann J. D. The Bacillus subtilis sigma D-dependent operon encoding the flagellar proteins FliD, FliS, and FliT. J Bacteriol. 1994 Jun;176(11):3093–3101. doi: 10.1128/jb.176.11.3093-3101.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Davidson B. E., MacDougall J., Saint Girons I. Physical map of the linear chromosome of the bacterium Borrelia burgdorferi 212, a causative agent of Lyme disease, and localization of rRNA genes. J Bacteriol. 1992 Jun;174(11):3766–3774. doi: 10.1128/jb.174.11.3766-3774.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Dingwall A., Garman J. D., Shapiro L. Organization and ordered expression of Caulobacter genes encoding flagellar basal body rod and ring proteins. J Mol Biol. 1992 Dec 20;228(4):1147–1162. doi: 10.1016/0022-2836(92)90322-b. [DOI] [PubMed] [Google Scholar]
  17. Fan F., Macnab R. M. Enzymatic characterization of FliI. An ATPase involved in flagellar assembly in Salmonella typhimurium. J Biol Chem. 1996 Dec 13;271(50):31981–31988. doi: 10.1074/jbc.271.50.31981. [DOI] [PubMed] [Google Scholar]
  18. Fredrick K. L., Helmann J. D. Dual chemotaxis signaling pathways in Bacillus subtilis: a sigma D-dependent gene encodes a novel protein with both CheW and CheY homologous domains. J Bacteriol. 1994 May;176(9):2727–2735. doi: 10.1128/jb.176.9.2727-2735.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fredrick K., Caramori T., Chen Y. F., Galizzi A., Helmann J. D. Promoter architecture in the flagellar regulon of Bacillus subtilis: high-level expression of flagellin by the sigma D RNA polymerase requires an upstream promoter element. Proc Natl Acad Sci U S A. 1995 Mar 28;92(7):2582–2586. doi: 10.1073/pnas.92.7.2582. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gabe J. D., Chang R. J., Slomiany R., Andrews W. H., McCaman M. T. Isolation of extracytoplasmic proteins from Serpulina hyodysenteriae B204 and molecular cloning of the flaB1 gene encoding a 38-kilodalton flagellar protein. Infect Immun. 1995 Jan;63(1):142–148. doi: 10.1128/iai.63.1.142-148.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ge Y., Charon N. W. An unexpected flaA homolog is present and expressed in Borrelia burgdorferi. J Bacteriol. 1997 Jan;179(2):552–556. doi: 10.1128/jb.179.2.552-556.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ge Y., Old I., Saint Girons I., Yelton D. B., Charon N. W. FliH and fliI of Borrelia burgdorferi are similar to flagellar and virulence factor export proteins of other bacteria. Gene. 1996 Feb 2;168(1):73–75. doi: 10.1016/0378-1119(95)00743-1. [DOI] [PubMed] [Google Scholar]
  23. Goldstein S. F., Buttle K. F., Charon N. W. Structural analysis of the Leptospiraceae and Borrelia burgdorferi by high-voltage electron microscopy. J Bacteriol. 1996 Nov;178(22):6539–6545. doi: 10.1128/jb.178.22.6539-6545.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Goldstein S. F., Charon N. W., Kreiling J. A. Borrelia burgdorferi swims with a planar waveform similar to that of eukaryotic flagella. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3433–3437. doi: 10.1073/pnas.91.8.3433. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Goldstein S. F., Charon N. W. Motility of the spirochete Leptospira. Cell Motil Cytoskeleton. 1988;9(2):101–110. doi: 10.1002/cm.970090202. [DOI] [PubMed] [Google Scholar]
  26. Goulbourne E. A., Jr, Greenberg E. P. Chemotaxis of Spirochaeta aurantia: involvement of membrane potential in chemosensory signal transduction. J Bacteriol. 1981 Dec;148(3):837–844. doi: 10.1128/jb.148.3.837-844.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Hardham J. M., Frye J. G., Stamm L. V. Identification and sequences of the Treponema pallidum fliM', fliY, fliP, fliQ, fliR and flhB' genes. Gene. 1995 Dec 1;166(1):57–64. doi: 10.1016/0378-1119(95)00583-x. [DOI] [PubMed] [Google Scholar]
  28. Harley C. B., Reynolds R. P. Analysis of E. coli promoter sequences. Nucleic Acids Res. 1987 Mar 11;15(5):2343–2361. doi: 10.1093/nar/15.5.2343. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Helmann J. D. Alternative sigma factors and the regulation of flagellar gene expression. Mol Microbiol. 1991 Dec;5(12):2875–2882. doi: 10.1111/j.1365-2958.1991.tb01847.x. [DOI] [PubMed] [Google Scholar]
  30. Hovind-Hougen K. Ultrastructure of spirochetes isolated from Ixodes ricinus and Ixodes dammini. Yale J Biol Med. 1984 Jul-Aug;57(4):543–548. [PMC free article] [PubMed] [Google Scholar]
  31. Iino T., Komeda Y., Kutsukake K., Macnab R. M., Matsumura P., Parkinson J. S., Simon M. I., Yamaguchi S. New unified nomenclature for the flagellar genes of Escherichia coli and Salmonella typhimurium. Microbiol Rev. 1988 Dec;52(4):533–535. doi: 10.1128/mr.52.4.533-535.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Isaacs R. D., Radolf J. D. Expression in Escherichia coli of the 37-kilodalton endoflagellar sheath protein of Treponema pallidum by use of the polymerase chain reaction and a T7 expression system. Infect Immun. 1990 Jul;58(7):2025–2034. doi: 10.1128/iai.58.7.2025-2034.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Jwang B., Dewing P., Fikrig E., Flavell R. A. The hook protein of Borrelia burgdorferi, encoded by the flgE gene, is serologically recognized in Lyme disease. Clin Diagn Lab Immunol. 1995 Sep;2(5):609–615. doi: 10.1128/cdli.2.5.609-615.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Kimsey R. B., Spielman A. Motility of Lyme disease spirochetes in fluids as viscous as the extracellular matrix. J Infect Dis. 1990 Nov;162(5):1205–1208. doi: 10.1093/infdis/162.5.1205. [DOI] [PubMed] [Google Scholar]
  35. Koopman M. B., de Leeuw O. S., van der Zeijst B. M., Kusters J. G. Cloning and DNA sequence analysis of a Serpulina (Treponema) hyodysenteriae gene encoding a periplasmic flagellar sheath protein. Infect Immun. 1992 Jul;60(7):2920–2925. doi: 10.1128/iai.60.7.2920-2925.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Kutsukake K., Iino T. Role of the FliA-FlgM regulatory system on the transcriptional control of the flagellar regulon and flagellar formation in Salmonella typhimurium. J Bacteriol. 1994 Jun;176(12):3598–3605. doi: 10.1128/jb.176.12.3598-3605.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Leschine S. B., Canale-Parola E. Rifampin as a selective agent for isolation of oral spirochetes. J Clin Microbiol. 1980 Dec;12(6):792–795. doi: 10.1128/jcm.12.6.792-795.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Limberger R. J., Slivienski L. L., El-Afandi M. C., Dantuono L. A. Organization, transcription, and expression of the 5' region of the fla operon of Treponema phagedenis and Treponema pallidum. J Bacteriol. 1996 Aug;178(15):4628–4634. doi: 10.1128/jb.178.15.4628-4634.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Limberger R. J., Slivienski L. L., Samsonoff W. A. Genetic and biochemical analysis of the flagellar hook of Treponema phagedenis. J Bacteriol. 1994 Jun;176(12):3631–3637. doi: 10.1128/jb.176.12.3631-3637.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Limberger R. J., Slivienski L. L., Yelton D. B., Charon N. W. Molecular genetic analysis of a class B periplasmic-flagellum gene of Treponema phagedenis. J Bacteriol. 1992 Oct;174(20):6404–6410. doi: 10.1128/jb.174.20.6404-6410.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Magnarelli L. A. Current status of laboratory diagnosis for Lyme disease. Am J Med. 1995 Apr 24;98(4A):10S–14S. doi: 10.1016/s0002-9343(99)80039-4. [DOI] [PubMed] [Google Scholar]
  42. Mirel D. B., Lauer P., Chamberlin M. J. Identification of flagellar synthesis regulatory and structural genes in a sigma D-dependent operon of Bacillus subtilis. J Bacteriol. 1994 Aug;176(15):4492–4500. doi: 10.1128/jb.176.15.4492-4500.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Mirel D. B., Lustre V. M., Chamberlin M. J. An operon of Bacillus subtilis motility genes transcribed by the sigma D form of RNA polymerase. J Bacteriol. 1992 Jul;174(13):4197–4204. doi: 10.1128/jb.174.13.4197-4204.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Noppa L., Burman N., Sadziene A., Barbour A. G., Bergström S. Expression of the flagellin gene in Borrelia is controlled by an alternative sigma factor. Microbiology. 1995 Jan;141(Pt 1):85–93. doi: 10.1099/00221287-141-1-85. [DOI] [PubMed] [Google Scholar]
  45. Ohnishi K., Ohto Y., Aizawa S., Macnab R. M., Iino T. FlgD is a scaffolding protein needed for flagellar hook assembly in Salmonella typhimurium. J Bacteriol. 1994 Apr;176(8):2272–2281. doi: 10.1128/jb.176.8.2272-2281.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Old I. G., MacDougall J., Saint Girons I., Davidson B. E. Mapping of genes on the linear chromosome of the bacterium Borrelia burgdorferi: possible locations for its origin of replication. FEMS Microbiol Lett. 1992 Dec 1;78(2-3):245–250. doi: 10.1016/0378-1097(92)90034-l. [DOI] [PubMed] [Google Scholar]
  47. Ordal G. W., Nettleton D. O., Hoch J. A. Genetics of Bacillus subtilis chemotaxis: isolation and mapping of mutations and cloning of chemotaxis genes. J Bacteriol. 1983 Jun;154(3):1088–1097. doi: 10.1128/jb.154.3.1088-1097.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Pallesen L., Hindersson P. Cloning and sequencing of a Treponema pallidum gene encoding a 31.3-kilodalton endoflagellar subunit (FlaB2). Infect Immun. 1989 Jul;57(7):2166–2172. doi: 10.1128/iai.57.7.2166-2172.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Parales J., Jr, Greenberg E. P. Analysis of the Spirochaeta aurantia flaA gene and transcript. FEMS Microbiol Lett. 1993 Feb 1;106(3):245–251. doi: 10.1111/j.1574-6968.1993.tb05971.x. [DOI] [PubMed] [Google Scholar]
  50. Postic D., Edlinger C., Richaud C., Grimont F., Dufresne Y., Perolat P., Baranton G., Grimont P. A. Two genomic species in Borrelia burgdorferi. Res Microbiol. 1990 May;141(4):465–475. doi: 10.1016/0923-2508(90)90072-x. [DOI] [PubMed] [Google Scholar]
  51. Quon K. C., Marczynski G. T., Shapiro L. Cell cycle control by an essential bacterial two-component signal transduction protein. Cell. 1996 Jan 12;84(1):83–93. doi: 10.1016/s0092-8674(00)80995-2. [DOI] [PubMed] [Google Scholar]
  52. Roberts E. D., Bohm R. P., Jr, Cogswell F. B., Lanners H. N., Lowrie R. C., Jr, Povinelli L., Piesman J., Philipp M. T. Chronic lyme disease in the rhesus monkey. Lab Invest. 1995 Feb;72(2):146–160. [PubMed] [Google Scholar]
  53. Rohrwild M., Coux O., Huang H. C., Moerschell R. P., Yoo S. J., Seol J. H., Chung C. H., Goldberg A. L. HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome. Proc Natl Acad Sci U S A. 1996 Jun 11;93(12):5808–5813. doi: 10.1073/pnas.93.12.5808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Roman S. J., Frantz B. B., Matsumura P. Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG. Gene. 1993 Oct 29;133(1):103–108. doi: 10.1016/0378-1119(93)90232-r. [DOI] [PubMed] [Google Scholar]
  55. Rosey E. L., Kennedy M. J., Petrella D. K., Ulrich R. G., Yancey R. J., Jr Inactivation of Serpulina hyodysenteriae flaA1 and flaB1 periplasmic flagellar genes by electroporation-mediated allelic exchange. J Bacteriol. 1995 Oct;177(20):5959–5970. doi: 10.1128/jb.177.20.5959-5970.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Rosey E. L., Kennedy M. J., Yancey R. J., Jr Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery. Infect Immun. 1996 Oct;64(10):4154–4162. doi: 10.1128/iai.64.10.4154-4162.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sadziene A., Thomas D. D., Bundoc V. G., Holt S. C., Barbour A. G. A flagella-less mutant of Borrelia burgdorferi. Structural, molecular, and in vitro functional characterization. J Clin Invest. 1991 Jul;88(1):82–92. doi: 10.1172/JCI115308. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. 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]
  59. Shapiro L. The bacterial flagellum: from genetic network to complex architecture. Cell. 1995 Feb 24;80(4):525–527. doi: 10.1016/0092-8674(95)90505-7. [DOI] [PubMed] [Google Scholar]
  60. Vogler A. P., Homma M., Irikura V. M., Macnab R. M. Salmonella typhimurium mutants defective in flagellar filament regrowth and sequence similarity of FliI to F0F1, vacuolar, and archaebacterial ATPase subunits. J Bacteriol. 1991 Jun;173(11):3564–3572. doi: 10.1128/jb.173.11.3564-3572.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Wallich R., Moter S. E., Simon M. M., Ebnet K., Heiberger A., Kramer M. D. The Borrelia burgdorferi flagellum-associated 41-kilodalton antigen (flagellin): molecular cloning, expression, and amplification of the gene. Infect Immun. 1990 Jun;58(6):1711–1719. doi: 10.1128/iai.58.6.1711-1719.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Zuberi A. R., Ying C., Bischoff D. S., Ordal G. W. Gene-protein relationships in the flagellar hook-basal body complex of Bacillus subtilis: sequences of the flgB, flgC, flgG, fliE and fliF genes. Gene. 1991 May 15;101(1):23–31. doi: 10.1016/0378-1119(91)90220-6. [DOI] [PubMed] [Google Scholar]

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

RESOURCES