Advertisement

Assaying Promoter Activity Using LacZ and GFP as Reporters

  • Paul Carroll
  • Jade James
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 465)

Abstract

The ability of bacteria to survive in a variety of different niches is due, in part, to their ability to respond and adapt to the environment. Extracellular signals are recognized by bacilli, and their responses are generally conducted at the transcript level. RNA polymerases recognize specific promoter regions on the genome and initiate transcription. Therefore, the analysis of gene expression is paramount to understanding the biology of an organism. In the case of pathogens, gene expression can alter during the course of the infection, and, therefore, specific targets can be identified for drug development. Promoter activity can be determined by cloning a promoter sequence upstream of a reporter gene and assaying the reporter activity, either from whole cells or from cell lysates. This chapter describes two reporter systems (GFP and LacZ) used for determining promoter activity that have been widely used in mycobacteria.

Keywords

β-galactosidase cell-free extract fluorescence GFP LacZ live bacilli mycobacteria reporter vector 

References

  1. 1.
    Sherman, D. R., Voskuil, M., Schnappinger, D., Liao, R., Harrell, M. I. & Schoolnik, G. K. (2001). Regulation of the Mycobacterium tuberculosis hypoxic response gene encoding alpha -crystallin. Proc Natl Acad Sci U S A. 98, 7534–9.PubMedCrossRefGoogle Scholar
  2. 2.
    Betts, J. C., Lukey, P. T., Robb, L. C., McAdam, R. A. & Duncan, K. (2002). Evaluation of a nutrient starvation model of Mycobacterium tuberculosis persistence by gene and protein expression profiling. Mol Microbiol. 43, 717–31.PubMedCrossRefGoogle Scholar
  3. 3.
    Fisher, M. A., Plikaytis, B. B. & Shinnick, T. M. (2002). Microarray analysis of the Mycobacterium tuberculosis transcriptional response to the acidic conditions found in phagosomes. J Bacteriol. 184, 4025–32.PubMedCrossRefGoogle Scholar
  4. 4.
    Talaat, A. M., Lyons, R., Howard, S. T. & Johnston, S. A. (2004). The temporal expression profile of Mycobacterium tuberculosis infection in mice. Proc Natl Acad Sci U S A. 101, 4602–7.PubMedCrossRefGoogle Scholar
  5. 5.
    Labidi, A., David, H. L. & Roulland-Dussoix, D. (1985). Restriction endonuclease mapping and cloning of Mycobacterium fortuitum var. fortuitum plasmid pAL5000. Ann Inst Pasteur Microbiol. 136B, 209–15.PubMedCrossRefGoogle Scholar
  6. 6.
    Snapper, S. B., Melton, R. E., Mustafa, S., Kieser, T. & Jacobs, W. R. Jr. (1990). Isolation and characterization of efficient plasmid transformation mutants of Mycobacterium smegmatis. Mol Microbiol. 4, 1911–9.PubMedCrossRefGoogle Scholar
  7. 7.
    Hinshelwood, S. & Stoker, N. G. (1992). An Escherichia coli-Mycobacterium shuttle cosmid vector, pMSC1. Gene. 110, 115–8.PubMedCrossRefGoogle Scholar
  8. 8.
    Guilhot, C., Gicquel, B. & Martin, C. (1992). Temperature-sensitive mutants of the Mycobacterium plasmid pAL5000. FEMS Microbiol Lett. 77, 181–6.PubMedCrossRefGoogle Scholar
  9. 9.
    Gicquel-Sanzey, B., Moniz-Pereira, J., Gheorghiu, M. & Rauzier, J. (1989). Structure of pAL5000, a plasmid from M. fortuitum and its utilization in transformation of mycobacteria. Acta Leprol. 7, 208–11.PubMedGoogle Scholar
  10. 10.
    Coons, A. H. & Kaplan, M. H. (1950). Localization of antigen in tissue cells; improvements in a method for the detection of antigen by means of fluorescent antibody. J Exp Med. 91, 1–13.PubMedCrossRefGoogle Scholar
  11. 11.
    Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. & Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science. 263, 802–5.PubMedCrossRefGoogle Scholar
  12. 12.
    Atkins, D. & Izant, J. G. (1995). Expression and analysis of the green fluorescent protein gene in the fission yeast Schizosaccharomyces pombe. Curr Genet. 28, 585–8.CrossRefGoogle Scholar
  13. 13.
    Lim, C. R., Kimata, Y., Oka, M., Nomaguchi, K. & Kohno, K. (1995). Thermosensitivity of green fluorescent protein fluorescence utilized to reveal novel nuclear-like compartments in a mutant nucleoporin NSP1. J Biochem (Tokyo). 118, 13–7.Google Scholar
  14. 14.
    Yeh, E., Gustafson, K. & Boulianne, G. L. (1995). Green fluorescent protein as a vital marker and reporter of gene expression in Drosophila. Proc Natl Acad Sci U S A. 92, 7036–40.PubMedCrossRefGoogle Scholar
  15. 15.
    Amsterdam, A., Lin, S. & Hopkins, N. (1995). The Aequorea victoria green fluorescent protein can be used as a reporter in live zebrafish embryos. Dev Biol. 171, 123–9.PubMedCrossRefGoogle Scholar
  16. 16.
    Rizzuto, R., Brini, M., Pizzo, P., Murgia, M. & Pozzan, T. (1995). Chimeric green fluorescent protein as a tool for visualizing subcellular organelles in living cells. Curr Biol. 5, 635–42.PubMedCrossRefGoogle Scholar
  17. 17.
    Ikawa, M., Kominami, K., Yoshimura, Y., Tanaka, K., Nishimune, Y. & Okabe, M. (1995). A rapid and non-invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP). FEBS Lett. 375, 125–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Hansen, M. C., Palmer, R. J. Jr., Udsen, C., White, D. C. & Molin, S. (2001). Assessment of GFP fluorescence in cells of Streptococcus gordonii under conditions of low pH and low oxygen concentration. Microbiology. 147, 1383–91.PubMedGoogle Scholar
  19. 19.
    Reichel, C., Mathur, J., Eckes, P., Langenkemper, K., Koncz, C., Schell, J., Reiss, B. & Maas, C. (1996). Enhanced green fluorescence by the expression of an Aequorea victoria green fluorescent protein mutant in mono- and dicotyledonous plant cells. Proc Natl Acad Sci U S A. 93, 5888–93.PubMedCrossRefGoogle Scholar
  20. 20.
    Daabrowski, S., Brillowska, A. & Kur, J. (1999). Use of the green fluorescent protein variant (YFP) to monitor MetArg human proinsulin production in Escherichia coli. Protein Expr Purif. 16, 315–23.PubMedCrossRefGoogle Scholar
  21. 21.
    Matz, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L. & Lukyanov, S. A. (1999). Fluorescent proteins from nonbioluminescent Anthozoa species. Nat Biotechnol. 17, 969–73.CrossRefGoogle Scholar
  22. 22.
    Triccas, J. A., Pinto, R. & Britton, W. J. (2002). Destabilized green fluorescent protein for monitoring transient changes in mycobacterial gene expression. Res Microbiol. 153, 379–83.PubMedCrossRefGoogle Scholar
  23. 23.
    Blokpoel, M. C., O'Toole, R., Smeulders, M. J. & Williams, H. D. (2003). Development and application of unstable GFP variants to kinetic studies of mycobacterial gene expression. J Microbiol Methods. 54, 203–11.PubMedCrossRefGoogle Scholar
  24. 24.
    Dulebohn, D. P., Cho, H. J. & Karzai, A. W. (2006). Role of conserved surface amino acids in binding of SmpB protein to SsrA RNA. J Biol Chem. 281, 28536–45.PubMedCrossRefGoogle Scholar
  25. 25.
    Jain, V., Sujatha, S., Ojha, A. K. & Chatterji, D. (2005). Identification and characterization of rel promoter element of Mycobacterium tuberculosis. Gene 351, 149–57.PubMedCrossRefGoogle Scholar
  26. 26.
    Haydel, S. E., Benjamin, W. H., Jr., Dunlap, N. E. & Clark-Curtiss, J. E. (2002). Expression, autoregulation, and DNA binding properties of the Mycobacterium tuberculosis TrcR response regulator. J Bacteriol. 184, 2192–203.PubMedCrossRefGoogle Scholar
  27. 27.
    Parish, T., Turner, J. & Stoker, N. G. (2001). amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis. BMC Microbiol. 1, 19.PubMedCrossRefGoogle Scholar
  28. 28.
    Kumar, D., Srivastava, B. S. & Srivastava, R. (1998). Genetic rearrangements leading to disruption of heterologous gene expression in mycobacteria: an observation with Escherichia coli beta-galactosidase in Mycobacterium smegmatis and its implication in vaccine development. Vaccine 16, 1212–5.PubMedCrossRefGoogle Scholar
  29. 29.
    Srivastava, R., Kumar, D., Subramaniam, P. & Srivastava, B. S. (1997). beta-Galactosidase reporter system in mycobacteria and its application in rapid antimycobacterial drug screening. Biochem Biophys Res Commun. 235, 602–5.PubMedCrossRefGoogle Scholar
  30. 30.
    Dhandayuthapani, S., Via, L. E., Thomas, C. A., Horowitz, P. M., Deretic, D. & Deretic, V. (1995). Green fluorescent protein as a marker for gene expression and cell biology of mycobacterial interactions with macrophages. Mol Microbiol. 17, 901–12.PubMedCrossRefGoogle Scholar
  31. 31.
    Via, L. E., Curcic, R., Mudd, M. H., Dhandayuthapani, S., Ulmer, R. J. & Deretic, V. (1996). Elements of signal transduction in Mycobacterium tuberculosis: in vitro phosphorylation and in vivo expression of the response regulator MtrA. J Bacteriol. 178, 3314–21.PubMedGoogle Scholar
  32. 32.
    Zahrt, T. C. & Deretic, V. (2000). An essential two-component signal transduction system in Mycobacterium tuberculosis. J Bacteriol. 182, 3832–8.PubMedCrossRefGoogle Scholar
  33. 33.
    O'Toole, R., Smeulders, M. J., Blokpoel, M. C., Kay, E. J., Lougheed, K. & Williams, H. D. (2003). A two-component regulator of universal stress protein expression and adaptation to oxygen starvation in Mycobacterium smegmatis. J Bacteriol. 185, 1543–54.PubMedCrossRefGoogle Scholar
  34. 34.
    Roberts, E. A., Clark, A., McBeth, S. & Friedman, R. L. (2004). Molecular characterization of the eis promoter of Mycobacterium tuberculosis. J Bacteriol. 186, 5410–7.PubMedCrossRefGoogle Scholar
  35. 35.
    Danelishvili, L., Poort, M. J. & Bermudez, L. E. (2004). Identification of Mycobacterium avium genes up-regulated in cultured macrophages and in mice. FEMS Microbiol Lett. 239, 41–9.PubMedCrossRefGoogle Scholar
  36. 36.
    Cowley, S. C. & Av-Gay, Y. (2001). Monitoring promoter activity and protein localization in Mycobacterium spp. using green fluorescent protein. Gene. 264, 225–31.PubMedCrossRefGoogle Scholar
  37. 37.
    Barletta, R. G., Snapper, B., Cirillo, J. D., Connell, N. D., Kim, D. D., Jacobs, W. R. & Bloom, B. R. (1990). Recombinant BCG as a candidate oral vaccine vector. Res Microbiol. 141, 931–9.PubMedCrossRefGoogle Scholar
  38. 38.
    Timm, J., Lim, E. M. & Gicquel, B. (1994). Escherichia coli-mycobacteria shuttle vectors for operon and gene fusions to lacZ: the pJEM series. J Bacteriol. 176, 6749–53.PubMedGoogle Scholar
  39. 39.
    Timm, J., Perilli, M. G., Duez, C., Trias, J., Orefici, G., Fattorini, L., Amicosante, G., Oratore, A., Joris, B., Frere, J. M., et al. (1994). Transcription and expression analysis, using lacZ and phoA gene fusions, of Mycobacterium fortuitum beta-lactamase genes cloned from a natural isolate and a high-level beta-lactamase producer. Mol Microbiol. 12, 491–504.PubMedCrossRefGoogle Scholar
  40. 40.
    Dellagostin, O. A., Esposito, G., Eales, L. J., Dale, J. W. & McFadden, J. (1995). Activity of mycobacterial promoters during intracellular and extracellular growth. Microbiology. 141, 1785–92.PubMedCrossRefGoogle Scholar
  41. 41.
    Ainsa, J. A., Martin, C., Cabeza, M., De la Cruz, F. & Mendiola, M. V. (1996). Construction of a family of Mycobacterium/Escherichia coli shuttle vectors derived from pAL5000 and pACYC184: their use for cloning an antibiotic-resistance gene from Mycobacterium fortuitum. Gene. 176, 23–6.PubMedCrossRefGoogle Scholar
  42. 42.
    Hotter, G. S., Wilson, T. & Collins, D. M. (2001). Identification of a cadmium-induced gene in Mycobacterium bovis and Mycobacterium tuberculosis. FEMS Microbiol Lett. 200, 151–5.PubMedCrossRefGoogle Scholar
  43. 43.
    Curcic, R., Dhandayuthapani, S. & Deretic, V. (1994). Gene expression in mycobacteria: transcriptional fusions based on xylE and analysis of the promoter region of the response regulator mtrA from Mycobacterium tuberculosis. Mol Microbiol. 13, 1057–64.PubMedCrossRefGoogle Scholar
  44. 44.
    Das Gupta, S. K., Bashyam, M. D. & Tyagi, A. K. (1993). Cloning and assessment of mycobacterial promoters by using a plasmid shuttle vector. J Bacteriol. 175, 5186–92.PubMedGoogle Scholar
  45. 45.
    Sarkis, G. J., Jacobs, W. R., Jr. & Hatfull, G. F. (1995). L5 luciferase reporter mycobacteriophages: a sensitive tool for the detection and assay of live mycobacteria. Mol Microbiol. 15, 1055–67.PubMedCrossRefGoogle Scholar
  46. 46.
    Blokpoel, M. C., Murphy, H. N., O'Toole, R., Wiles, S., Runn, E. S., Stewart, G. R., Young, D. B. & Robertson, B. D. (2005). Tetracycline-inducible gene regulation in mycobacteria. Nucleic Acids Res. 33, e22.PubMedCrossRefGoogle Scholar
  47. 47.
    Dussurget, O., Timm, J., Gomez, M., Gold, B., Yu, S. W., Sabol, S. Z., Holmes, R. K., Jacobs, W. R. & Smith, I. (1999). Transcriptional control of the iron-responsive fxbA gene by the mycobacterial regulator IdeR. J Bacteriol. 181, 3402–3408.PubMedGoogle Scholar
  48. 48.
    Miller, J. H. (1972). Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  49. 49.
    Miller, J. H. (1972). Assay of beta-galactosidase activity. In Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar
  50. 50.
    Brown, A. C. & Parish, T. (2006). Instability of the acetamide-inducible expression vector pJAM2 in Mycobacterium tuberculosis. Plasmid. 55, 81–6.PubMedCrossRefGoogle Scholar
  51. 51.
    Chawla, M. & Das Gupta, S. K. (1999). Transposition-induced structural instability of Escherichia coli-mycobacteria shuttle vectors. Plasmid. 41, 135–40.PubMedCrossRefGoogle Scholar
  52. 52.
    Medeiros, M. A., Dellagostin, O. A., Armoa, G. R., Degrave, W. M., De Mendonca-Lima, L., Lopes, M. Q., Costa, J. F., McFadden, J. & McIntosh, D. (2002). Comparative evaluation of Mycobacterium vaccae as a surrogate cloning host for use in the study of mycobacterial genetics. Microbiology. 148, 1999–2009.PubMedGoogle Scholar
  53. 53.
    Springer, B., Sander, P., Sedlacek, L., Ellrott, K. & Bottger, E. C. (2001). Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors. Int J Med Microbiol. 290, 669–75.PubMedCrossRefGoogle Scholar
  54. 54.
    Chan Kwo Chion, C. K., Askew, S. E. & Leak, D. J. (2005). Cloning, expression, and site-directed mutagenesis of the propene monooxygenase genes from Mycobacterium sp. strain M156. Appl Environ Microbiol. 71, 1909–14.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  1. 1.Institute of Cell and Molecular Science, Barts and the London, Queen Mary’s School of Medicine and DentistryLondonUK
  2. 2.Centre for Infectious Disease, Institute of Cell and Molecular ScienceBarts and the London, Queen Mary’s School of Medicine and DentistryLondonUK

Personalised recommendations