Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1995 Feb;61(2):660–668. doi: 10.1128/aem.61.2.660-668.1995

Expression vectors for the use of eukaryotic luciferases as bacterial markers with different colors of luminescence.

A Cebolla 1, M E Vázquez 1, A J Palomares 1
PMCID: PMC167327  PMID: 7574604

Abstract

An easy way to identify microorganisms is to provide them with gene markers that confer a unique phenotype. Several genetic constructions were developed to use eukaryotic luciferase genes for bacterial tagging. The firefly and click bettle luciferase genes, luc and lucOR, respectively, were cloned under constitutive control and regulated control from different transcriptional units driven by P1, lambda PR, and Ptrc promoters. Comparison of the expression of each gene in Escherichia coli cells from identical promoters showed that bioluminescence produced by luc could be detected luminometrically in a more sensitive manner. In contrast, luminescence from intact lucOR-expressing cells was much more stable and resistant to high temperatures than that from luc-expressing cells. To analyze the behavior of these constructions in other gram-negative bacteria, gene fusions with luc genes were cloned on broad-host-range vectors. Measurements of light emission from Rhizobium meliloti, Agrobacterium tumefaciens, and Pseudomonas putida cells indicated that both luciferases were poorly expressed from P1 in most bacterial hosts. In contrast, the lambda promoter PR yielded constitutively high levels of luciferase expression in all bacterial species tested. PR activity was not regulated by temperature when the thermosensitive repressor cI857 was present in the bacterial species tested, except for E. coli. In contrast, the regulated lacIq-Ptrc::lucOR fusion expression system behaved in a manner similar to that observed in E. coli cells. After IPTG (isopropyl-beta-D-thiogalactopyranoside) induction, this system produced the highest levels of lucOR expression in all bacterial species tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (624.9 KB).

Selected References

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

  1. Amann E., Brosius J. "ATG vectors' for regulated high-level expression of cloned genes in Escherichia coli. Gene. 1985;40(2-3):183–190. doi: 10.1016/0378-1119(85)90041-1. [DOI] [PubMed] [Google Scholar]
  2. Bagdasarian M. M., Amann E., Lurz R., Rückert B., Bagdasarian M. Activity of the hybrid trp-lac (tac) promoter of Escherichia coli in Pseudomonas putida. Construction of broad-host-range, controlled-expression vectors. Gene. 1983 Dec;26(2-3):273–282. doi: 10.1016/0378-1119(83)90197-x. [DOI] [PubMed] [Google Scholar]
  3. Bej A. K., Mahbubani M. H., Atlas R. M. Amplification of nucleic acids by polymerase chain reaction (PCR) and other methods and their applications. Crit Rev Biochem Mol Biol. 1991;26(3-4):301–334. doi: 10.3109/10409239109114071. [DOI] [PubMed] [Google Scholar]
  4. Boyer H. W., Roulland-Dussoix D. A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol. 1969 May 14;41(3):459–472. doi: 10.1016/0022-2836(69)90288-5. [DOI] [PubMed] [Google Scholar]
  5. Breitling R., Sorokin A. V., Behnke D. Temperature-inducible gene expression in Bacillus subtilis mediated by the cI857-encoded repressor of bacteriophage lambda. Gene. 1990 Sep 1;93(1):35–40. doi: 10.1016/0378-1119(90)90132-b. [DOI] [PubMed] [Google Scholar]
  6. Cebolla A., Ruiz-Berraquero F., Palomares A. J. Expression and quantification of firefly luciferase under control of Rhizobium meliloti symbiotic promoters. J Biolumin Chemilumin. 1991 Jul-Sep;6(3):177–184. doi: 10.1002/bio.1170060307. [DOI] [PubMed] [Google Scholar]
  7. Cebolla A., Ruiz-Berraquero F., Palomares A. J. Stable Tagging of Rhizobium meliloti with the Firefly Luciferase Gene for Environmental Monitoring. Appl Environ Microbiol. 1993 Aug;59(8):2511–2519. doi: 10.1128/aem.59.8.2511-2519.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  9. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Enrique Vázquez M., Cebolla A., Palomares A. J. Controlled expression of click beetle luciferase using a bacterial operator-repressor system. FEMS Microbiol Lett. 1994 Aug 1;121(1):11–18. doi: 10.1111/j.1574-6968.1994.tb07068.x. [DOI] [PubMed] [Google Scholar]
  11. Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
  12. Fürste J. P., Pansegrau W., Frank R., Blöcker H., Scholz P., Bagdasarian M., Lanka E. Molecular cloning of the plasmid RP4 primase region in a multi-host-range tacP expression vector. Gene. 1986;48(1):119–131. doi: 10.1016/0378-1119(86)90358-6. [DOI] [PubMed] [Google Scholar]
  13. Herrero M., de Lorenzo V., Timmis K. N. Transposon vectors containing non-antibiotic resistance selection markers for cloning and stable chromosomal insertion of foreign genes in gram-negative bacteria. J Bacteriol. 1990 Nov;172(11):6557–6567. doi: 10.1128/jb.172.11.6557-6567.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Jacobs W. R., Jr, Barletta R. G., Udani R., Chan J., Kalkut G., Sosne G., Kieser T., Sarkis G. J., Hatfull G. F., Bloom B. R. Rapid assessment of drug susceptibilities of Mycobacterium tuberculosis by means of luciferase reporter phages. Science. 1993 May 7;260(5109):819–822. doi: 10.1126/science.8484123. [DOI] [PubMed] [Google Scholar]
  15. Klock G., Unger B., Gatz C., Hillen W., Altenbuchner J., Schmid K., Schmitt R. Heterologous repressor-operator recognition among four classes of tetracycline resistance determinants. J Bacteriol. 1985 Jan;161(1):326–332. doi: 10.1128/jb.161.1.326-332.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Lampinen J., Koivisto L., Wahlsten M., Mäntsälä P., Karp M. Expression of luciferase genes from different origins in Bacillus subtilis. Mol Gen Genet. 1992 Apr;232(3):498–504. doi: 10.1007/BF00266255. [DOI] [PubMed] [Google Scholar]
  17. Legocki R. P., Legocki M., Baldwin T. O., Szalay A. A. Bioluminescence in soybean root nodules: Demonstration of a general approach to assay gene expression in vivo by using bacterial luciferase. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9080–9084. doi: 10.1073/pnas.83.23.9080. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Palomares A. J., DeLuca M. A., Helinski D. R. Firefly luciferase as a reporter enzyme for measuring gene expression in vegetative and symbiotic Rhizobium meliloti and other gram-negative bacteria. Gene. 1989 Sep 1;81(1):55–64. doi: 10.1016/0378-1119(89)90336-3. [DOI] [PubMed] [Google Scholar]
  20. Peschke U., Beuck V., Bujard H., Gentz R., Le Grice S. Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis. J Mol Biol. 1985 Dec 5;186(3):547–555. doi: 10.1016/0022-2836(85)90129-9. [DOI] [PubMed] [Google Scholar]
  21. Schmidhauser T. J., Helinski D. R. Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria. J Bacteriol. 1985 Oct;164(1):446–455. doi: 10.1128/jb.164.1.446-455.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shaw J. J., Dane F., Geiger D., Kloepper J. W. Use of bioluminescence for detection of genetically engineered microorganisms released into the environment. Appl Environ Microbiol. 1992 Jan;58(1):267–273. doi: 10.1128/aem.58.1.267-273.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Stewart G. S., Williams P. lux genes and the applications of bacterial bioluminescence. J Gen Microbiol. 1992 Jul;138(7):1289–1300. doi: 10.1099/00221287-138-7-1289. [DOI] [PubMed] [Google Scholar]
  24. Winstanley C., Morgan J. A., Pickup R. W., Jones J. G., Saunders J. R. Differential regulation of lambda pL and pR promoters by a cI repressor in a broad-host-range thermoregulated plasmid marker system. Appl Environ Microbiol. 1989 Apr;55(4):771–777. doi: 10.1128/aem.55.4.771-777.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Wood K. V., DeLuca M. Photographic detection of luminescence in Escherichia coli containing the gene for firefly luciferase. Anal Biochem. 1987 Mar;161(2):501–507. doi: 10.1016/0003-2697(87)90480-5. [DOI] [PubMed] [Google Scholar]
  26. Wood K. V., Lam Y. A., McElroy W. D. Introduction to beetle luciferases and their applications. J Biolumin Chemilumin. 1989 Jul;4(1):289–301. doi: 10.1002/bio.1170040141. [DOI] [PubMed] [Google Scholar]
  27. Wood K. V., Lam Y. A., McElroy W. D., Seliger H. H. Bioluminescent click beetles revisited. J Biolumin Chemilumin. 1989 Jul;4(1):31–39. doi: 10.1002/bio.1170040110. [DOI] [PubMed] [Google Scholar]
  28. Wood K. V., Lam Y. A., Seliger H. H., McElroy W. D. Complementary DNA coding click beetle luciferases can elicit bioluminescence of different colors. Science. 1989 May 12;244(4905):700–702. doi: 10.1126/science.2655091. [DOI] [PubMed] [Google Scholar]
  29. de Lorenzo V., Herrero M., Jakubzik U., Timmis K. N. Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. J Bacteriol. 1990 Nov;172(11):6568–6572. doi: 10.1128/jb.172.11.6568-6572.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. de Wet J. R., Wood K. V., DeLuca M., Helinski D. R., Subramani S. Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol. 1987 Feb;7(2):725–737. doi: 10.1128/mcb.7.2.725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. de Wet J. R., Wood K. V., Helinski D. R., DeLuca M. Cloning of firefly luciferase cDNA and the expression of active luciferase in Escherichia coli. Proc Natl Acad Sci U S A. 1985 Dec;82(23):7870–7873. doi: 10.1073/pnas.82.23.7870. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES