Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Feb;62(2):403–407. doi: 10.1128/aem.62.2.403-407.1996

Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P-450 in Rhodococcus strains.

Z Q Shao 1, R Behki 1
PMCID: PMC167811  PMID: 8593046

Abstract

A cytochrome P-450 system in Rhodococcus strains, encoded by thcB, thcC, and thcD, participates in the degradation of thiocarbamates and several other pesticides. The regulation of the system was investigated by fusing a truncated lacZ in frame to thcB, the structural gene for the cytochrome P-450 monooxygenase. Analysis of the thcB-lacZ fusion showed that the expression of thcB was 10-fold higher in the presence of the herbicide EPTC (s-ethyl dipropylthiocarbamate). Similar enhancement of the thcB-lacZ expression was found with other thiocarbamate pesticides. Atrazine, simazine, or carbofuran, although metabolized by the system, had no effect on the thcB-lacZ expression. The presence of glucose slightly increased the expression of thcB-lacZ, indicating no catabolic repression of the thcB-lacZ expression. The expression of thcB-lacZ was decreased more than twofold in Luria-Bertani medium. This was due in part to cysteine, which repressed thcB-lacZ expression. It was confirmed that the thcR gene, which is transcribed divergently from thcB, codes for a positive regulatory protein which is essential for the thcB-lacZ expression. Studies of the thcR-lacZ protein fusion showed that the thcR gene is expressed constitutively.

Full Text

The Full Text of this article is available as a PDF (218.1 KB).

Selected References

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

  1. Behki R., Topp E., Dick W., Germon P. Metabolism of the herbicide atrazine by Rhodococcus strains. Appl Environ Microbiol. 1993 Jun;59(6):1955–1959. doi: 10.1128/aem.59.6.1955-1959.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Denome S. A., Olson E. S., Young K. D. Identification and Cloning of Genes Involved in Specific Desulfurization of Dibenzothiophene by Rhodococcus sp. Strain IGTS8. Appl Environ Microbiol. 1993 Sep;59(9):2837–2843. doi: 10.1128/aem.59.9.2837-2843.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Desomer J., Dhaese P., Montagu M. V. Transformation of Rhodococcus fascians by High-Voltage Electroporation and Development of R. fascians Cloning Vectors. Appl Environ Microbiol. 1990 Sep;56(9):2818–2825. doi: 10.1128/aem.56.9.2818-2825.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Eltis L. D., Karlson U., Timmis K. N. Purification and characterization of cytochrome P450RR1 from Rhodococcus rhodochrous. Eur J Biochem. 1993 Apr 1;213(1):211–216. doi: 10.1111/j.1432-1033.1993.tb17750.x. [DOI] [PubMed] [Google Scholar]
  5. Finnerty W. R. The biology and genetics of the genus Rhodococcus. Annu Rev Microbiol. 1992;46:193–218. doi: 10.1146/annurev.mi.46.100192.001205. [DOI] [PubMed] [Google Scholar]
  6. Gallegos M. T., Michán C., Ramos J. L. The XylS/AraC family of regulators. Nucleic Acids Res. 1993 Feb 25;21(4):807–810. doi: 10.1093/nar/21.4.807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Guengerich F. P. Enzymatic oxidation of xenobiotic chemicals. Crit Rev Biochem Mol Biol. 1990;25(2):97–153. doi: 10.3109/10409239009090607. [DOI] [PubMed] [Google Scholar]
  8. Hashimoto Y., Nishiyama M., Ikehata O., Horinouchi S., Beppu T. Cloning and characterization of an amidase gene from Rhodococcus species N-774 and its expression in Escherichia coli. Biochim Biophys Acta. 1991 Feb 16;1088(2):225–233. doi: 10.1016/0167-4781(91)90058-t. [DOI] [PubMed] [Google Scholar]
  9. Hashimoto Y., Nishiyama M., Yu F., Watanabe I., Horinouchi S., Beppu T. Development of a host-vector system in a Rhodococcus strain and its use for expression of the cloned nitrile hydratase gene cluster. J Gen Microbiol. 1992 May;138(5):1003–1010. doi: 10.1099/00221287-138-5-1003. [DOI] [PubMed] [Google Scholar]
  10. Hill R., Hart S., Illing N., Kirby R., Woods D. R. Cloning and expression of Rhodococcus genes encoding pigment production in Escherichia coli. J Gen Microbiol. 1989 Jun;135(6):1507–1513. doi: 10.1099/00221287-135-6-1507. [DOI] [PubMed] [Google Scholar]
  11. Ikehata O., Nishiyama M., Horinouchi S., Beppu T. Primary structure of nitrile hydratase deduced from the nucleotide sequence of a Rhodococcus species and its expression in Escherichia coli. Eur J Biochem. 1989 May 15;181(3):563–570. doi: 10.1111/j.1432-1033.1989.tb14761.x. [DOI] [PubMed] [Google Scholar]
  12. Karlson U., Dwyer D. F., Hooper S. W., Moore E. R., Timmis K. N., Eltis L. D. Two independently regulated cytochromes P-450 in a Rhodococcus rhodochrous strain that degrades 2-ethoxyphenol and 4-methoxybenzoate. J Bacteriol. 1993 Mar;175(5):1467–1474. doi: 10.1128/jb.175.5.1467-1474.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Nagy I., Compernolle F., Ghys K., Vanderleyden J., De Mot R. A single cytochrome P-450 system is involved in degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine by Rhodococcus sp. strain NI86/21. Appl Environ Microbiol. 1995 May;61(5):2056–2060. doi: 10.1128/aem.61.5.2056-2060.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Nagy I., Schoofs G., Compernolle F., Proost P., Vanderleyden J., de Mot R. Degradation of the thiocarbamate herbicide EPTC (S-ethyl dipropylcarbamothioate) and biosafening by Rhodococcus sp. strain NI86/21 involve an inducible cytochrome P-450 system and aldehyde dehydrogenase. J Bacteriol. 1995 Feb;177(3):676–687. doi: 10.1128/jb.177.3.676-687.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. O'Keefe D. P., Harder P. A. Occurrence and biological function of cytochrome P450 monooxygenases in the actinomycetes. Mol Microbiol. 1991 Sep;5(9):2099–2105. doi: 10.1111/j.1365-2958.1991.tb02139.x. [DOI] [PubMed] [Google Scholar]
  16. Okazaki O., Guengerich F. P. Evidence for specific base catalysis in N-dealkylation reactions catalyzed by cytochrome P450 and chloroperoxidase. Differences in rates of deprotonation of aminium radicals as an explanation for high kinetic hydrogen isotope effects observed with peroxidases. J Biol Chem. 1993 Jan 25;268(3):1546–1552. [PubMed] [Google Scholar]
  17. Patel N. V., Omer C. A. Phenobarbital and sulfonylurea-inducible operons encoding herbicide metabolizing cytochromes P-450 in Streptomyces griseolus. Gene. 1992 Mar 1;112(1):67–76. doi: 10.1016/0378-1119(92)90304-8. [DOI] [PubMed] [Google Scholar]
  18. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Sariaslani F. S. Microbial cytochromes P-450 and xenobiotic metabolism. Adv Appl Microbiol. 1991;36:133–178. doi: 10.1016/s0065-2164(08)70453-2. [DOI] [PubMed] [Google Scholar]
  20. Shao Z. Q., Behki R. Cloning of the genes for degradation of the herbicides EPTC (S-ethyl dipropylthiocarbamate) and atrazine from Rhodococcus sp. strain TE1. Appl Environ Microbiol. 1995 May;61(5):2061–2065. doi: 10.1128/aem.61.5.2061-2065.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Shao Z. Q., Seffens W., Mulbry W., Behki R. M. Cloning and expression of the s-triazine hydrolase gene (trzA) from Rhodococcus corallinus and development of Rhodococcus recombinant strains capable of dealkylating and dechlorinating the herbicide atrazine. J Bacteriol. 1995 Oct;177(20):5748–5755. doi: 10.1128/jb.177.20.5748-5755.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Shao Z., Dick W. A., Behki R. M. An improved Escherichia coli-Rhodococcus shuttle vector and plasmid transformation in Rhodococcus spp. using electroporation. Lett Appl Microbiol. 1995 Oct;21(4):261–266. doi: 10.1111/j.1472-765x.1995.tb01056.x. [DOI] [PubMed] [Google Scholar]
  23. Shapira S. K., Chou J., Richaud F. V., Casadaban M. J. New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused to lacZ gene sequences encoding an enzymatically active carboxy-terminal portion of beta-galactosidase. Gene. 1983 Nov;25(1):71–82. doi: 10.1016/0378-1119(83)90169-5. [DOI] [PubMed] [Google Scholar]
  24. Tam A. C., Behki R. M., Khan S. U. Isolation and characterization of an s-ethyl-N,N-dipropylthiocarbamate-degrading Arthrobacter strain and evidence for plasmid-associated s-ethyl-N,N-dipropylthiocarbamate degradation. Appl Environ Microbiol. 1987 May;53(5):1088–1093. doi: 10.1128/aem.53.5.1088-1093.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Warhurst A. M., Fewson C. A. Biotransformations catalyzed by the genus Rhodococcus. Crit Rev Biotechnol. 1994;14(1):29–73. doi: 10.3109/07388559409079833. [DOI] [PubMed] [Google Scholar]

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

RESOURCES