1887

Abstract

regulates genes in response to a number of environmental signals such as temperature and pH. A green fluorescent protein (GFP) reporter system using the , and promoters from B31 was introduced into infectious clonal isolates of strains B31 and N40 to monitor and compare gene expression in response to pH and temperature . GFP could be assayed by epifluorescence microscopy, immunoblotting or spectrofluorometry and was an accurate reporter of target gene expression. It was determined that only 179 bp 5′ of was sufficient to regulate the reporter in response to pH and temperature in B31. The loss of linear plasmid (lp) 25, lp28-1, lp36 and lp56 had no effect on the ability of B31 to regulate in response to pH or temperature. The amount of OspC in N40 transformants was unaffected by changes in pH or temperature of the culture medium. This suggests that regulation of gene expression in response to pH and temperature may vary between these two strains.

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2003-07-01
2019-12-08
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References

  1. Albano, C. R., Randers-Eichhorn, L., Chang, Q., Bently, W. E. & Rao, G. ( 1996; ). Quantitative measurement of green fluorescent protein expression. Biotechnol Techniques 10, 953–958.
    [Google Scholar]
  2. Babb, K., El-Hage, N., Miller, J. C., Carroll, J. A. & Stevenson, B. ( 2001; ). Distinct regulatory pathways control expression of Borrelia burgdorferi infection-associated OspC and Erp surface proteins. Infect Immun 69, 4146–4153.[CrossRef]
    [Google Scholar]
  3. Barthold, S. W., Moody, K. D., Terwilliger, G. A., Duray, P. H., Jacoby, R. O. & Steere, A. C. ( 1988; ). Experimental Lyme arthritis in rats infected with Borrelia burgdorferi. J Infect Dis 157, 842–846.[CrossRef]
    [Google Scholar]
  4. Barthold, S. W., de Souza, M. S., Janotka, J. L., Smith, A. L. & Persing, D. H. ( 1993; ). Chronic Lyme borreliosis in the laboratory mouse. Am J Pathol 143, 959–971.
    [Google Scholar]
  5. Bono, J. L., Elias, A. F., Kupko, J. J., 3rd, Stevenson, B., Tilly, K. & Rosa, P. ( 2000; ). Efficient targeted mutagenesis in Borrelia burgdorferi. J Bacteriol 182, 2445–2452.[CrossRef]
    [Google Scholar]
  6. Carroll, J. A. & Gherardini, F. C. ( 1996; ). Membrane protein variations associated with in vitro passage of Borrelia burgdorferi. Infect Immun 64, 392–398.
    [Google Scholar]
  7. Carroll, J. A., Garon, C. F. & Schwan, T. G. ( 1999; ). Effects of environmental pH on membrane proteins in Borrelia burgdorferi. Infect Immun 67, 3181–3187.
    [Google Scholar]
  8. Carroll, J. A., Cordova, R. M. & Garon, C. F. ( 2000; ). Identification of 11 pH-regulated genes in Borrelia burgdorferi localizing to linear plasmids. Infect Immun 68, 6677–6684.[CrossRef]
    [Google Scholar]
  9. Carroll, J. A., El-Hage, N., Miller, J. C., Babb, K. & Stevenson, B. ( 2001; ). Borrelia burgdorferi RevA antigen is a surface-exposed outer membrane protein whose expression is regulated in response to environmental temperature and pH. Infect Immun 69, 5286–5293.[CrossRef]
    [Google Scholar]
  10. Cassatt, D. R., Patel, N. K., Ulbrandt, N. D. & Hanson, M. S. ( 1998; ). DbpA, but not OspA, is expressed by Borrelia burgdorferi during spirochetemia and is a target for protective antibodies. Infect Immun 66, 5379–5387.
    [Google Scholar]
  11. Cormack, B. P., Valdivia, R. H. & Falkow, S. ( 1996; ). FACS-optimized mutants of the green fluorescent protein (GFP). Gene 173, 33–38.[CrossRef]
    [Google Scholar]
  12. Crameri, A., Whitehorn, E. A., Tate, E. & Stemmer, W. P. ( 1996; ). Improved green fluorescent protein by molecular evolution using DNA shuffling. Nat Biotechnol 14, 315–319.[CrossRef]
    [Google Scholar]
  13. Eggers, C. H., Caimano, M. J., Clawson, M. L., Miller, W. G., Samuels, D. S. & Radolf, J. D. ( 2002; ). Identification of loci critical for replication and compatibility of a Borrelia burgdorferi cp32 plasmid and use of a cp32-based shuttle vector for expression of fluorescent reporters in the Lyme disease spirochete. Mol Microbiol 42, 281–295.
    [Google Scholar]
  14. Elias, A. F., Stewart, P. E., Grimm, D. & 8 other authors ( 2002; ). Clonal polymorphism of Borrelia burgdorferi B31 MI: implications for mutagenesis in an infectious strain background. Infect Immun 70, 2139–2150.[CrossRef]
    [Google Scholar]
  15. Fikrig, E., Feng, W., Aversa, J., Schoen, R. T. & Flavell, R. A. ( 1998; ). Differential expression of Borrelia burgdorferi genes during erythema migrans and Lyme arthritis. J Infect Dis 178, 1198–1201.[CrossRef]
    [Google Scholar]
  16. Fraser, C. M., Casjens, S., Huang, W. M. & 35 other authors ( 1997; ). Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi. Nature 390, 580–586.[CrossRef]
    [Google Scholar]
  17. Frye, J. G., Kremer, B. K., Hoover, T. R. & Gherardini, F. C. ( 1999; ). Preliminary characterization of rpoS and changes in protein expression induced by stationary phase in Borrelia burgdorferi. In 99th General Meeting of the American Society for Microbiology, May 30–June 3, Chicago, IL, USA, Abstract DIB-259.
  18. Indest, K. J., Ramamoorthy, R., Sole, M., Gilmore, R. D., Johnson, B. J. & Philipp, M. T. ( 1997; ). Cell-density-dependent expression of Borrelia burgdorferi lipoproteins in vitro. Infect Immun 65, 1165–1171.
    [Google Scholar]
  19. Lu, C., Albano, C. R., Bentley, W. E. & Rao, G. ( 2002; ). Differential rates of gene expression monitored by green fluorescent protein. Biotechnol Bioeng 79, 429–437.[CrossRef]
    [Google Scholar]
  20. McDowell, J. V., Sung, S. Y., Labandeira-Rey, M., Skare, J. T. & Marconi, R. T. ( 2001; ). Analysis of mechanisms associated with loss of infectivity of clonal populations of Borrelia burgdorferi B31MI. Infect Immun 69, 3670–3677.[CrossRef]
    [Google Scholar]
  21. Ohnishi, J., Piesman, J. & de Silva, A. M. ( 2001; ). Antigenic and genetic heterogeneity of Borrelia burgdorferi populations transmitted by ticks. Proc Natl Acad Sci U S A 98, 670–675.[CrossRef]
    [Google Scholar]
  22. Patterson, G. H., Knobel, S. M., Sharif, W. D., Kain, S. R. & Piston, D. W. ( 1997; ). Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy. Biophys J 73, 2782–2790.[CrossRef]
    [Google Scholar]
  23. Purser, J. E. & Norris, S. J. ( 2000; ). Correlation between plasmid content and infectivity in Borrelia burgdorferi. Proc Natl Acad Sci U S A 97, 13865–13870.[CrossRef]
    [Google Scholar]
  24. Ramamoorthy, R. & Philipp, M. T. ( 1998; ). Differential expression of Borrelia burgdorferi proteins during growth in vitro. Infect Immun 66, 5119–5124.
    [Google Scholar]
  25. Ramamoorthy, R. & Scholl-Meeker, D. ( 2001; ). Borrelia burgdorferi proteins whose expression is similarly affected by culture temperature and pH. Infect Immun 69, 2739–2742.[CrossRef]
    [Google Scholar]
  26. Samuels, D. S. ( 1995; ). Electrotransformation of the spirochete Borrelia burgdorferi. Methods Mol Biol 47, 253–259.
    [Google Scholar]
  27. Sartakova, M., Dobrikova, E. & Cabello, F. C. ( 2000; ). Development of an extrachromosomal cloning vector system for use in Borrelia burgdorferi. Proc Natl Acad Sci U S A 97, 4850–4855.[CrossRef]
    [Google Scholar]
  28. Schwan, T. G., Burgdorfer, W. & Garon, C. F. ( 1988; ). Changes in infectivity and plasmid profile of the Lyme disease spirochete, Borrelia burgdorferi, as a result of in vitro cultivation. Infect Immun 56, 1831–1836.
    [Google Scholar]
  29. Schwan, T. G., Piesman, J., Golde, W. T., Dolan, M. C. & Rosa, P. A. ( 1995; ). Induction of an outer surface protein on Borrelia burgdorferi during tick feeding. Proc Natl Acad Sci U S A 92, 2909–2913.[CrossRef]
    [Google Scholar]
  30. Sohaskey, C. D., Arnold, C. & Barbour, A. G. ( 1997; ). Analysis of promoters in Borrelia burgdorferi by use of a transiently expressed reporter gene. J Bacteriol 179, 6837–6842.
    [Google Scholar]
  31. Sohaskey, C. D., Zuckert, W. R. & Barbour, A. G. ( 1999; ). The extended promoters for two outer membrane lipoprotein genes of Borrelia spp. uniquely include a T-rich region. Mol Microbiol 33, 41–51.[CrossRef]
    [Google Scholar]
  32. Stevenson, B., Schwan, T. G. & Rosa, P. A. ( 1995; ). Temperature-related differential expression of antigens in the Lyme disease spirochete, Borrelia burgdorferi. Infect Immun 63, 4535–4539.
    [Google Scholar]
  33. Stewart, P. E., Thalken, R., Bono, J. L. & Rosa, P. ( 2001; ). Isolation of a circular plasmid region sufficient for autonomous replication and transformation of infectious Borrelia burgdorferi. Mol Microbiol 39, 714–721.[CrossRef]
    [Google Scholar]
  34. Thomas, V., Anguita, J., Samanta, S., Rosa, P. A., Stewart, P., Barthold, S. W. & Fikrig, E. ( 2001; ). Dissociation of infectivity and pathogenicity in Borrelia burgdorferi. Infect Immun 69, 3507–3509.[CrossRef]
    [Google Scholar]
  35. Towbin, H., Staehelin, T. & Gordon, J. ( 1979; ). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A 76, 4350–4354.[CrossRef]
    [Google Scholar]
  36. Tsien, R. Y. ( 1998; ). The green fluorescent protein. Annu Rev Biochem 67, 509–544.[CrossRef]
    [Google Scholar]
  37. Xu, Y., Kodner, C., Coleman, L. & Johnson, R. C. ( 1996; ). Correlation of plasmids with infectivity of Borrelia burgdorferi sensu stricto type strain B31. Infect Immun 64, 3870–3876.
    [Google Scholar]
  38. Yang, X., Goldberg, M. S., Popova, T. G., Schoeler, G. B., Wikel, S. K., Hagman, K. E. & Norgard, M. V. ( 2000; ). Interdependence of environmental factors influencing reciprocal patterns of gene expression in virulent Borrelia burgdorferi. Mol Microbiol 37, 1470–1479.[CrossRef]
    [Google Scholar]
  39. Yokoe, H. & Meyer, T. ( 1996; ). Spatial dynamics of GFP-tagged proteins investigated by local fluorescence enhancement. Nat Biotechnol 14, 1252–1256.[CrossRef]
    [Google Scholar]
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