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. 1994 Apr;176(8):2293–2299. doi: 10.1128/jb.176.8.2293-2299.1994

Intracellular generation of superoxide as a by-product of Vibrio harveyi luciferase expressed in Escherichia coli.

B González-Flecha 1, B Demple 1
PMCID: PMC205351  PMID: 8157597

Abstract

Luciferase genes are widely used as reporters of gene expression because of the high sensitivity of chemiluminescence detection and the possibility of monitoring light production in intact cells. We engineered fusions of the Escherichia coli soxS promoter to the luciferase structural genes (luxAB) from Vibrio harveyi. Since soxS transcription is positively triggered by the activated SoxR protein in response to agents such as paraquat that generate intracellular superoxide, we hoped to use this construct as a sensitive reporter of redox stress agents. Although a soxR+ soxS'::luxAB fusion exhibited a paraquat-inducible synthesis of luciferase, a smaller increase was consistently observed even in the absence of known soxRS inducers. This endogenous induction was soxR dependent and was further characterized by introducing a plasmid carrying the luciferase structural genes without the soxS promoter into a strain carrying a soxS'::lacZ fusion in the bacterial chromosome. These cells exhibited increased beta-galactosidase expression as they grew into mid-log phase. This increase was ascribed to luciferase activity because beta-galactosidase induction was suppressed (but not eliminated) when the substrate n-decanal was present in the medium. The soxS'::luxAB plasmid transformed superoxide dismutase-deficient strains very poorly under aerobic conditions but just as efficiently as a control plasmid under anaerobic conditions. The production of hydrogen peroxide, the dismutation product of superoxide anion, was significantly increased in strains carrying bacterial luciferase and maximal in the absence of n-decanal. Taken collectively, these data point to the generation of significant amounts of intracellular superoxide by bacterial luciferase, the possible mechanism of which is discussed. In addition to providing insights into the role of superoxide in the activation of the SoxR protein, these results suggest caution in the interpretation of experiments using luciferase as a reporter of gene expression.

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Selected References

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

  1. Alam J., Cook J. L. Reporter genes: application to the study of mammalian gene transcription. Anal Biochem. 1990 Aug 1;188(2):245–254. doi: 10.1016/0003-2697(90)90601-5. [DOI] [PubMed] [Google Scholar]
  2. Amábile-Cuevas C. F., Demple B. Molecular characterization of the soxRS genes of Escherichia coli: two genes control a superoxide stress regulon. Nucleic Acids Res. 1991 Aug 25;19(16):4479–4484. doi: 10.1093/nar/19.16.4479. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baldwin T. O., Berends T., Bunch T. A., Holzman T. F., Rausch S. K., Shamansky L., Treat M. L., Ziegler M. M. Cloning of the luciferase structural genes from Vibrio harveyi and expression of bioluminescence in Escherichia coli. Biochemistry. 1984 Jul 31;23(16):3663–3667. doi: 10.1021/bi00311a014. [DOI] [PubMed] [Google Scholar]
  4. Bolivar F., Rodriguez R. L., Betlach M. C., Boyer H. W. Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene. 1977;2(2):75–93. doi: 10.1016/0378-1119(77)90074-9. [DOI] [PubMed] [Google Scholar]
  5. Boylan M., Pelletier J., Meighen E. A. Fused bacterial luciferase subunits catalyze light emission in eukaryotes and prokaryotes. J Biol Chem. 1989 Feb 5;264(4):1915–1918. [PubMed] [Google Scholar]
  6. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  7. Brunmark A., Cadenas E. Redox and addition chemistry of quinoid compounds and its biological implications. Free Radic Biol Med. 1989;7(4):435–477. doi: 10.1016/0891-5849(89)90126-3. [DOI] [PubMed] [Google Scholar]
  8. Bustamante J., Guerra L., Bredeston L., Mordoh J., Boveris A. Melanin content and hydroperoxide metabolism in human melanoma cells. Exp Cell Res. 1991 Oct;196(2):172–176. doi: 10.1016/0014-4827(91)90247-r. [DOI] [PubMed] [Google Scholar]
  9. Carlioz A., Touati D. Isolation of superoxide dismutase mutants in Escherichia coli: is superoxide dismutase necessary for aerobic life? EMBO J. 1986 Mar;5(3):623–630. doi: 10.1002/j.1460-2075.1986.tb04256.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Carmi O. A., Stewart G. S., Ulitzur S., Kuhn J. Use of bacterial luciferase to establish a promoter probe vehicle capable of nondestructive real-time analysis of gene expression in Bacillus spp. J Bacteriol. 1987 May;169(5):2165–2170. doi: 10.1128/jb.169.5.2165-2170.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Chance B., Sies H., Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979 Jul;59(3):527–605. doi: 10.1152/physrev.1979.59.3.527. [DOI] [PubMed] [Google Scholar]
  12. Christman M. F., Morgan R. W., Jacobson F. S., Ames B. N. Positive control of a regulon for defenses against oxidative stress and some heat-shock proteins in Salmonella typhimurium. Cell. 1985 Jul;41(3):753–762. doi: 10.1016/s0092-8674(85)80056-8. [DOI] [PubMed] [Google Scholar]
  13. Claiborne A., Fridovich I. Purification of the o-dianisidine peroxidase from Escherichia coli B. Physicochemical characterization and analysis of its dual catalatic and peroxidatic activities. J Biol Chem. 1979 May 25;254(10):4245–4252. [PubMed] [Google Scholar]
  14. Condee C. W., Summers A. O. A mer-lux transcriptional fusion for real-time examination of in vivo gene expression kinetics and promoter response to altered superhelicity. J Bacteriol. 1992 Dec;174(24):8094–8101. doi: 10.1128/jb.174.24.8094-8101.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Demple B., Amábile-Cuevas C. F. Redox redux: the control of oxidative stress responses. Cell. 1991 Nov 29;67(5):837–839. doi: 10.1016/0092-8674(91)90355-3. [DOI] [PubMed] [Google Scholar]
  16. Demple B., Halbrook J. Inducible repair of oxidative DNA damage in Escherichia coli. Nature. 1983 Aug 4;304(5925):466–468. doi: 10.1038/304466a0. [DOI] [PubMed] [Google Scholar]
  17. Engebrecht J., Simon M., Silverman M. Measuring gene expression with light. Science. 1985 Mar 15;227(4692):1345–1347. doi: 10.1126/science.2983423. [DOI] [PubMed] [Google Scholar]
  18. Escher A., O'Kane D. J., Lee J., Szalay A. A. Bacterial luciferase alpha beta fusion protein is fully active as a monomer and highly sensitive in vivo to elevated temperature. Proc Natl Acad Sci U S A. 1989 Sep;86(17):6528–6532. doi: 10.1073/pnas.86.17.6528. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fraenkel D. G., Parola A. "Up-promoter" mutations of glucose 6-phosphate dehydrogenase in Escherichia coli. J Mol Biol. 1972 Oct 28;71(1):107–111. doi: 10.1016/0022-2836(72)90405-6. [DOI] [PubMed] [Google Scholar]
  20. Francis K. P., Gallagher M. P. Light emission from a Mudlux transcriptional fusion in Salmonella typhimurium is stimulated by hydrogen peroxide and by interaction with the mouse macrophage cell line J774.2. Infect Immun. 1993 Feb;61(2):640–649. doi: 10.1128/iai.61.2.640-649.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Giulivi C., Turrens J. F., Boveris A. Chemiluminescence enhancement by trypanocidal drugs and by inhibitors of antioxidant enzymes in Trypanosoma cruzi. Mol Biochem Parasitol. 1988 Sep;30(3):243–251. doi: 10.1016/0166-6851(88)90093-x. [DOI] [PubMed] [Google Scholar]
  22. González-Flecha B., Cutrin J. C., Boveris A. Time course and mechanism of oxidative stress and tissue damage in rat liver subjected to in vivo ischemia-reperfusion. J Clin Invest. 1993 Feb;91(2):456–464. doi: 10.1172/JCI116223. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Greenberg J. T., Monach P., Chou J. H., Josephy P. D., Demple B. Positive control of a global antioxidant defense regulon activated by superoxide-generating agents in Escherichia coli. Proc Natl Acad Sci U S A. 1990 Aug;87(16):6181–6185. doi: 10.1073/pnas.87.16.6181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. HASTINGS J. W., RILEY W. H., MASSA J. THE PURIFICATION PROPERTIES, AND CHEMILUMINESCENT QUANTUM YIELD OF BACTERIAL LUCIFERASE. J Biol Chem. 1965 Mar;240:1473–1481. [PubMed] [Google Scholar]
  25. Hassan H. M., Fridovich I. Regulation of the synthesis of catalase and peroxidase in Escherichia coli. J Biol Chem. 1978 Sep 25;253(18):6445–6420. [PubMed] [Google Scholar]
  26. Imlay J. A., Fridovich I. Assay of metabolic superoxide production in Escherichia coli. J Biol Chem. 1991 Apr 15;266(11):6957–6965. [PubMed] [Google Scholar]
  27. Kirchner G., Roberts J. L., Gustafson G. D., Ingolia T. D. Active bacterial luciferase from a fused gene: expression of a Vibrio harveyi luxAB translational fusion in bacteria, yeast and plant cells. Gene. 1989 Sep 30;81(2):349–354. doi: 10.1016/0378-1119(89)90195-9. [DOI] [PubMed] [Google Scholar]
  28. Kurfürst M., Ghisla S., Presswood R., Hastings J. W. Structure and catalytic inactivity of the bacterial luciferase neutral flavin radical. Eur J Biochem. 1982 Apr 1;123(2):355–361. doi: 10.1111/j.1432-1033.1982.tb19775.x. [DOI] [PubMed] [Google Scholar]
  29. Liochev S. I., Fridovich I. Fumarase C, the stable fumarase of Escherichia coli, is controlled by the soxRS regulon. Proc Natl Acad Sci U S A. 1992 Jul 1;89(13):5892–5896. doi: 10.1073/pnas.89.13.5892. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Misra H. P., Fridovich I. The univalent reduction of oxygen by reduced flavins and quinones. J Biol Chem. 1972 Jan 10;247(1):188–192. [PubMed] [Google Scholar]
  31. Nunoshiba T., Hidalgo E., Amábile Cuevas C. F., Demple B. Two-stage control of an oxidative stress regulon: the Escherichia coli SoxR protein triggers redox-inducible expression of the soxS regulatory gene. J Bacteriol. 1992 Oct;174(19):6054–6060. doi: 10.1128/jb.174.19.6054-6060.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nunoshiba T., deRojas-Walker T., Wishnok J. S., Tannenbaum S. R., Demple B. Activation by nitric oxide of an oxidative-stress response that defends Escherichia coli against activated macrophages. Proc Natl Acad Sci U S A. 1993 Nov 1;90(21):9993–9997. doi: 10.1073/pnas.90.21.9993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Olsson O., Escher A., Sandberg G., Schell J., Koncz C., Szalay A. A. Engineering of monomeric bacterial luciferases by fusion of luxA and luxB genes in Vibrio harveyi. Gene. 1989 Sep 30;81(2):335–347. doi: 10.1016/0378-1119(89)90194-7. [DOI] [PubMed] [Google Scholar]
  34. Pazzagli M., Devine J. H., Peterson D. O., Baldwin T. O. Use of bacterial and firefly luciferases as reporter genes in DEAE-dextran-mediated transfection of mammalian cells. Anal Biochem. 1992 Aug 1;204(2):315–323. doi: 10.1016/0003-2697(92)90245-3. [DOI] [PubMed] [Google Scholar]
  35. Puntarulo S., Sánchez R. A., Boveris A. Hydrogen peroxide metabolism in soybean embryonic axes at the onset of germination. Plant Physiol. 1988 Feb;86(2):626–630. doi: 10.1104/pp.86.2.626. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Tsaneva I. R., Weiss B. soxR, a locus governing a superoxide response regulon in Escherichia coli K-12. J Bacteriol. 1990 Aug;172(8):4197–4205. doi: 10.1128/jb.172.8.4197-4205.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Turrens J. F., Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980 Nov 1;191(2):421–427. doi: 10.1042/bj1910421. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Ulitzur S., Weiser I., Yannai S. A new, sensitive and simple bioluminescence test for mutagenic compounds. Mutat Res. 1980 Apr;74(2):113–124. doi: 10.1016/0165-1161(80)90237-x. [DOI] [PubMed] [Google Scholar]
  39. Wu J., Weiss B. Two divergently transcribed genes, soxR and soxS, control a superoxide response regulon of Escherichia coli. J Bacteriol. 1991 May;173(9):2864–2871. doi: 10.1128/jb.173.9.2864-2871.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wu J., Weiss B. Two-stage induction of the soxRS (superoxide response) regulon of Escherichia coli. J Bacteriol. 1992 Jun;174(12):3915–3920. doi: 10.1128/jb.174.12.3915-3920.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. 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]

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