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. 1996 Aug;62(8):2940–2946. doi: 10.1128/aem.62.8.2940-2946.1996

Identification of an alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase in Rhodococcus sp. strain RHA1 and cloning of the gene.

J E Hauschild 1, E Masai 1, K Sugiyama 1, T Hatta 1, K Kimbara 1, M Fukuda 1, K Yano 1
PMCID: PMC168081  PMID: 8702287

Abstract

Gram-positive Rhodococcus sp. strain RHA1 possesses strong polychlorinated biphenyl-degrading capabilities. An RHA1 bphC gene mutant, strain RDC1, had been previously constructed (E. Masai, A. Yamada, J. M. Healy, T. Hatta, K. Kimbara, M. Fukuda, and K. Yano, Appl. Environ. Microbiol. 61:2079-2085, 1995). An alternative 2,3-dihydroxybiphenyl 1,2-dioxygenase (2,3-DHBD), designated EtbC, was identified in RDC1 cells grown on ethylbenzene. EtbC contained the broadest substrate specificity of any meta cleavage dioxygenase identified in a Rhodococcus strain to date, including RHA1 BphC. EtbC was purified to near homogeneity from RDC1 cells grown on ethylbenzene, and a 58-amino-acid NH2-terminal sequence was determined. The NH2-terminal amino acid sequence was used for the identification of the etbC gene from an RDC1 chromosomal DNA 2,3-DHBD expression library. The etbC gene was successfully cloned, and we report here the determination of its nucleotide sequence. The substrate specificity patterns of cell extract and native nondenaturing polyacrylamide gel electrophoresis analysis identified the coexpression of two 2,3-DHBDs (BphC and EtbC) in RHA1 cells grown on either biphenyl or ethylbenzene. The possible implication of coexpressed BphC extradiol dioxygenases in the strong polychlorinated-biphenyl degradation activity of RHA1 was suggested.

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Selected References

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  1. Ahmed M., Focht D. D. Degradation of polychlorinated biphenyls by two species of Achromobacter. Can J Microbiol. 1973 Jan;19(1):47–52. doi: 10.1139/m73-007. [DOI] [PubMed] [Google Scholar]
  2. Arensdorf J. J., Focht D. D. Formation of chlorocatechol meta cleavage products by a pseudomonad during metabolism of monochlorobiphenyls. Appl Environ Microbiol. 1994 Aug;60(8):2884–2889. doi: 10.1128/aem.60.8.2884-2889.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Asturias J. A., Eltis L. D., Prucha M., Timmis K. N. Analysis of three 2,3-dihydroxybiphenyl 1,2-dioxygenases found in Rhodococcus globerulus P6. Identification of a new family of extradiol dioxygenases. J Biol Chem. 1994 Mar 11;269(10):7807–7815. [PubMed] [Google Scholar]
  4. Asturias J. A., Timmis K. N. Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes in the gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6. J Bacteriol. 1993 Aug;175(15):4631–4640. doi: 10.1128/jb.175.15.4631-4640.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bedard D. L., Haberl M. L., May R. J., Brennan M. J. Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H850. Appl Environ Microbiol. 1987 May;53(5):1103–1112. doi: 10.1128/aem.53.5.1103-1112.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bedard D. L., Unterman R., Bopp L. H., Brennan M. J., Haberl M. L., Johnson C. Rapid assay for screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls. Appl Environ Microbiol. 1986 Apr;51(4):761–768. doi: 10.1128/aem.51.4.761-768.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. 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]
  8. Candidus S., van Pée K. H., Lingens F. The catechol 2,3-dioxygenase gene of Rhodococcus rhodochrous CTM: nucleotide sequence, comparison with isofunctional dioxygenases and evidence for an active-site histidine. Microbiology. 1994 Feb;140(Pt 2):321–330. doi: 10.1099/13500872-140-2-321. [DOI] [PubMed] [Google Scholar]
  9. Eltis L. D., Hofmann B., Hecht H. J., Lünsdorf H., Timmis K. N. Purification and crystallization of 2,3-dihydroxybiphenyl 1,2-dioxygenase. J Biol Chem. 1993 Feb 5;268(4):2727–2732. [PubMed] [Google Scholar]
  10. Erickson B. D., Mondello F. J. Enhanced biodegradation of polychlorinated biphenyls after site-directed mutagenesis of a biphenyl dioxygenase gene. Appl Environ Microbiol. 1993 Nov;59(11):3858–3862. doi: 10.1128/aem.59.11.3858-3862.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Erickson B. D., Mondello F. J. Nucleotide sequencing and transcriptional mapping of the genes encoding biphenyl dioxygenase, a multicomponent polychlorinated-biphenyl-degrading enzyme in Pseudomonas strain LB400. J Bacteriol. 1992 May;174(9):2903–2912. doi: 10.1128/jb.174.9.2903-2912.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Furukawa K., Arimura N., Miyazaki T. Nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene of Pseudomonas pseudoalcaligenes. J Bacteriol. 1987 Jan;169(1):427–429. doi: 10.1128/jb.169.1.427-429.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Furukawa K., Arimura N. Purification and properties of 2,3-dihydroxybiphenyl dioxygenase from polychlorinated biphenyl-degrading Pseudomonas pseudoalcaligenes and Pseudomonas aeruginosa carrying the cloned bphC gene. J Bacteriol. 1987 Feb;169(2):924–927. doi: 10.1128/jb.169.2.924-927.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Furukawa K., Hirose J., Suyama A., Zaiki T., Hayashida S. Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon). J Bacteriol. 1993 Aug;175(16):5224–5232. doi: 10.1128/jb.175.16.5224-5232.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Furukawa K., Miyazaki T. Cloning of a gene cluster encoding biphenyl and chlorobiphenyl degradation in Pseudomonas pseudoalcaligenes. J Bacteriol. 1986 May;166(2):392–398. doi: 10.1128/jb.166.2.392-398.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Furukawa K., Tomizuka N., Kamibayashi A. Effect of chlorine substitution on the bacterial metabolism of various polychlorinated biphenyls. Appl Environ Microbiol. 1979 Aug;38(2):301–310. doi: 10.1128/aem.38.2.301-310.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ghosal D., You I. S., Gunsalus I. C. Nucleotide sequence and expression of gene nahH of plasmid NAH7 and homology with gene xylE of TOL pWWO. Gene. 1987;55(1):19–28. doi: 10.1016/0378-1119(87)90244-7. [DOI] [PubMed] [Google Scholar]
  18. Guo L. H., Yang R. C., Wu R. An improved strategy for rapid direct sequencing of both strands of long DNA molecules cloned in a plasmid. Nucleic Acids Res. 1983 Aug 25;11(16):5521–5540. doi: 10.1093/nar/11.16.5521. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Happe B., Eltis L. D., Poth H., Hedderich R., Timmis K. N. Characterization of 2,2',3-trihydroxybiphenyl dioxygenase, an extradiol dioxygenase from the dibenzofuran- and dibenzo-p-dioxin-degrading bacterium Sphingomonas sp. strain RW1. J Bacteriol. 1993 Nov;175(22):7313–7320. doi: 10.1128/jb.175.22.7313-7320.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Harayama S., Rekik M. Bacterial aromatic ring-cleavage enzymes are classified into two different gene families. J Biol Chem. 1989 Sep 15;264(26):15328–15333. [PubMed] [Google Scholar]
  21. Hayase N., Taira K., Furukawa K. Pseudomonas putida KF715 bphABCD operon encoding biphenyl and polychlorinated biphenyl degradation: cloning, analysis, and expression in soil bacteria. J Bacteriol. 1990 Feb;172(2):1160–1164. doi: 10.1128/jb.172.2.1160-1164.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hirose J., Suyama A., Hayashida S., Furukawa K. Construction of hybrid biphenyl (bph) and toluene (tod) genes for functional analysis of aromatic ring dioxygenases. Gene. 1994 Jan 28;138(1-2):27–33. doi: 10.1016/0378-1119(94)90779-x. [DOI] [PubMed] [Google Scholar]
  23. Kabisch M., Fortnagel P. Nucleotide sequence of the metapyrocatechase II (catechol 2,3-oxygenase II) gene mpcII from Alcaligenes eutrophus JMP 222. Nucleic Acids Res. 1990 Sep 25;18(18):5543–5543. doi: 10.1093/nar/18.18.5543. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kimbara K., Hashimoto T., Fukuda M., Koana T., Takagi M., Oishi M., Yano K. Cloning and sequencing of two tandem genes involved in degradation of 2,3-dihydroxybiphenyl to benzoic acid in the polychlorinated biphenyl-degrading soil bacterium Pseudomonas sp. strain KKS102. J Bacteriol. 1989 May;171(5):2740–2747. doi: 10.1128/jb.171.5.2740-2747.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Klecka G. M., Gibson D. T. Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol. Appl Environ Microbiol. 1981 May;41(5):1159–1165. doi: 10.1128/aem.41.5.1159-1165.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Kohler H. P., Kohler-Staub D., Focht D. D. Cometabolism of polychlorinated biphenyls: enhanced transformation of Aroclor 1254 by growing bacterial cells. Appl Environ Microbiol. 1988 Aug;54(8):1940–1945. doi: 10.1128/aem.54.8.1940-1945.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Kuhm A. E., Stolz A., Ngai K. L., Knackmuss H. J. Purification and characterization of a 1,2-dihydroxynaphthalene dioxygenase from a bacterium that degrades naphthalenesulfonic acids. J Bacteriol. 1991 Jun;173(12):3795–3802. doi: 10.1128/jb.173.12.3795-3802.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  29. Maeda M., Chung S. Y., Song E., Kudo T. Multiple genes encoding 2,3-dihydroxybiphenyl 1,2-dioxygenase in the gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus erythropolis TA421, isolated from a termite ecosystem. Appl Environ Microbiol. 1995 Feb;61(2):549–555. doi: 10.1128/aem.61.2.549-555.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Masai E., Yamada A., Healy J. M., Hatta T., Kimbara K., Fukuda M., Yano K. Characterization of biphenyl catabolic genes of gram-positive polychlorinated biphenyl degrader Rhodococcus sp. strain RHA1. Appl Environ Microbiol. 1995 Jun;61(6):2079–2085. doi: 10.1128/aem.61.6.2079-2085.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Nakai C., Kagamiyama H., Nozaki M., Nakazawa T., Inouye S., Ebina Y., Nakazawa A. Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2. J Biol Chem. 1983 Mar 10;258(5):2923–2928. [PubMed] [Google Scholar]
  32. Porzio M. A., Pearson A. M. Improved resolution of myofibrillar proteins with sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Biochim Biophys Acta. 1977 Jan 25;490(1):27–34. doi: 10.1016/0005-2795(77)90102-7. [DOI] [PubMed] [Google Scholar]
  33. Ramos J. L., Wasserfallen A., Rose K., Timmis K. N. Redesigning metabolic routes: manipulation of TOL plasmid pathway for catabolism of alkylbenzoates. Science. 1987 Jan 30;235(4788):593–596. doi: 10.1126/science.3468623. [DOI] [PubMed] [Google Scholar]
  34. 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]
  35. Seto M., Kimbara K., Shimura M., Hatta T., Fukuda M., Yano K. A Novel Transformation of Polychlorinated Biphenyls by Rhodococcus sp. Strain RHA1. Appl Environ Microbiol. 1995 Sep;61(9):3353–3358. doi: 10.1128/aem.61.9.3353-3358.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Seto M., Masai E., Ida M., Hatta T., Kimbara K., Fukuda M., Yano K. Multiple Polychlorinated Biphenyl Transformation Systems in the Gram-Positive Bacterium Rhodococcus sp. Strain RHA1. Appl Environ Microbiol. 1995 Dec;61(12):4510–4513. doi: 10.1128/aem.61.12.4510-4513.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Shingler V., Powlowski J., Marklund U. Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J Bacteriol. 1992 Feb;174(3):711–724. doi: 10.1128/jb.174.3.711-724.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Sondossi M., Sylvestre M., Ahmad D. Effects of chlorobenzoate transformation on the Pseudomonas testosteroni biphenyl and chlorobiphenyl degradation pathway. Appl Environ Microbiol. 1992 Feb;58(2):485–495. doi: 10.1128/aem.58.2.485-495.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Taira K., Hayase N., Arimura N., Yamashita S., Miyazaki T., Furukawa K. Cloning and nucleotide sequence of the 2,3-dihydroxybiphenyl dioxygenase gene from the PCB-degrading strain of Pseudomonas paucimobilis Q1. Biochemistry. 1988 May 31;27(11):3990–3996. doi: 10.1021/bi00411a015. [DOI] [PubMed] [Google Scholar]
  40. 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]
  41. Zylstra G. J., Gibson D. T. Toluene degradation by Pseudomonas putida F1. Nucleotide sequence of the todC1C2BADE genes and their expression in Escherichia coli. J Biol Chem. 1989 Sep 5;264(25):14940–14946. [PubMed] [Google Scholar]

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