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. 1997 Aug;179(15):4841–4849. doi: 10.1128/jb.179.15.4841-4849.1997

Cloning of genes involved in carbazole degradation of Pseudomonas sp. strain CA10: nucleotide sequences of genes and characterization of meta-cleavage enzymes and hydrolase.

S I Sato 1, N Ouchiyama 1, T Kimura 1, H Nojiri 1, H Yamane 1, T Omori 1
PMCID: PMC179332  PMID: 9244273

Abstract

The DNA fragment encoding meta-cleavage enzymes and the meta-cleavage compound hydrolase, involved in carbazole degradation, was cloned from the carbazole-utilizing bacterium Pseudomonas sp. strain CA10. DNA sequence analysis of this 2.6-kb SmaI-SphI fragment revealed that there were three open reading frames (ORF1, ORF2, and ORF3, in this gene order). ORF1 and ORF2 were indispensable for meta-cleavage activity for 2'-aminobiphenyl-2,3-diol and its easily available analog, 2,3-dihydroxybiphenyl, and were designated carBa and carBb, respectively. The alignment of CarBb with other meta-cleavage enzymes indicated that CarBb may have a non-heme iron cofactor coordinating site. On the basis of the phylogenetic tree, CarBb was classified as a member of the protocatechuate 4,5-dioxygenase family. This unique extradiol dioxygenase, CarB, had significantly higher affinity and about 20-times-higher meta-cleavage activity for 2,3-dihydroxybiphenyl than for catechol derivatives. The putative polypeptide encoded by ORF3 was homologous with meta-cleavage compound hydrolases in other bacteria, and ORF3 was designated carC. The hydrolase activity of CarC for 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid, the meta-cleavage compound of 2,3-dihydroxybiphenyl, was 40 times higher than that for 2-hydroxy-6-oxohepta-2,4-dienoic acid, the meta-cleavage compound of 3-methylcatechol. Alignment analysis and the phylogenetic tree indicate that CarC has greatest homologies with hydrolases involved in the monoaromatic compound degradation pathway. These results suggest the possibility that CarC is a novel type of hydrolase.

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Selected References

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

  1. Ahmad D., Fraser J., Sylvestre M., Larose A., Khan A., Bergeron J., Juteau J. M., Sondossi M. Sequence of the bphD gene encoding 2-hydroxy-6-oxo-(phenyl/chlorophenyl)hexa-2,4-dienoic acid (HOP/cPDA) hydrolase involved in the biphenyl/polychlorinated biphenyl degradation pathway in Comamonas testosteroni: evidence suggesting involvement of Ser112 in catalytic activity. Gene. 1995 Apr 14;156(1):69–74. doi: 10.1016/0378-1119(95)00073-f. [DOI] [PubMed] [Google Scholar]
  2. Arcos J. C., Argus M. F. Molecular geometry and carcinogenic activity of aromatic compounds. New perspectives. Adv Cancer Res. 1968;11:305–471. doi: 10.1016/s0065-230x(08)60390-5. [DOI] [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. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [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. Denome S. A., Oldfield C., Nash L. J., Young K. D. Characterization of the desulfurization genes from Rhodococcus sp. strain IGTS8. J Bacteriol. 1994 Nov;176(21):6707–6716. doi: 10.1128/jb.176.21.6707-6716.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eaton R. W., Timmis K. N. Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204. J Bacteriol. 1986 Oct;168(1):123–131. doi: 10.1128/jb.168.1.123-131.1986. [DOI] [PMC free article] [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. Ensley B. D., Ratzkin B. J., Osslund T. D., Simon M. J., Wackett L. P., Gibson D. T. Expression of naphthalene oxidation genes in Escherichia coli results in the biosynthesis of indigo. Science. 1983 Oct 14;222(4620):167–169. doi: 10.1126/science.6353574. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Grosser R. J., Warshawsky D., Vestal J. R. Indigenous and enhanced mineralization of pyrene, benzo[a]pyrene, and carbazole in soils. Appl Environ Microbiol. 1991 Dec;57(12):3462–3469. doi: 10.1128/aem.57.12.3462-3469.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Habe H., Kasuga K., Nojiri H., Yamane H., Omori T. Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01. Appl Environ Microbiol. 1996 Dec;62(12):4471–4477. doi: 10.1128/aem.62.12.4471-4477.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Han S., Eltis L. D., Timmis K. N., Muchmore S. W., Bolin J. T. Crystal structure of the biphenyl-cleaving extradiol dioxygenase from a PCB-degrading pseudomonad. Science. 1995 Nov 10;270(5238):976–980. doi: 10.1126/science.270.5238.976. [DOI] [PubMed] [Google Scholar]
  15. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Heiss G., Stolz A., Kuhm A. E., Müller C., Klein J., Altenbuchner J., Knackmuss H. J. Characterization of a 2,3-dihydroxybiphenyl dioxygenase from the naphthalenesulfonate-degrading bacterium strain BN6. J Bacteriol. 1995 Oct;177(20):5865–5871. doi: 10.1128/jb.177.20.5865-5871.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Hofer B., Eltis L. D., Dowling D. N., Timmis K. N. Genetic analysis of a Pseudomonas locus encoding a pathway for biphenyl/polychlorinated biphenyl degradation. Gene. 1993 Aug 16;130(1):47–55. doi: 10.1016/0378-1119(93)90345-4. [DOI] [PubMed] [Google Scholar]
  19. Horn J. M., Harayama S., Timmis K. N. DNA sequence determination of the TOL plasmid (pWWO) xylGFJ genes of Pseudomonas putida: implications for the evolution of aromatic catabolism. Mol Microbiol. 1991 Oct;5(10):2459–2474. doi: 10.1111/j.1365-2958.1991.tb02091.x. [DOI] [PubMed] [Google Scholar]
  20. Kabisch M., Fortnagel P. Nucleotide sequence of metapyrocatechase I (catechol 2,3-oxygenase I) gene mpcI from Alcaligenes eutrophus JMP222. Nucleic Acids Res. 1990 Jun 11;18(11):3405–3406. doi: 10.1093/nar/18.11.3405. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. 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]
  22. Kobayashi T., Kurane R., Nakajima K., Nakamura Y., Kirimura K., Usami S. Isolation of bacteria degrading carbazole under microaerobic conditions, i.e. nitrogen gas substituted conditions. Biosci Biotechnol Biochem. 1995 May;59(5):932–933. doi: 10.1271/bbb.59.932. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. 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]
  25. 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]
  26. Menn F. M., Zylstra G. J., Gibson D. T. Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1. Gene. 1991 Jul 31;104(1):91–94. doi: 10.1016/0378-1119(91)90470-v. [DOI] [PubMed] [Google Scholar]
  27. Noda Y., Nishikawa S., Shiozuka K., Kadokura H., Nakajima H., Yoda K., Katayama Y., Morohoshi N., Haraguchi T., Yamasaki M. Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis. J Bacteriol. 1990 May;172(5):2704–2709. doi: 10.1128/jb.172.5.2704-2709.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Nordlund I., Shingler V. Nucleotide sequences of the meta-cleavage pathway enzymes 2-hydroxymuconic semialdehyde dehydrogenase and 2-hydroxymuconic semialdehyde hydrolase from Pseudomonas CF600. Biochim Biophys Acta. 1990 Jun 21;1049(2):227–230. doi: 10.1016/0167-4781(90)90046-5. [DOI] [PubMed] [Google Scholar]
  29. Omori T., Monna L., Saiki Y., Kodama T. Desulfurization of dibenzothiophene by Corynebacterium sp. strain SY1. Appl Environ Microbiol. 1992 Mar;58(3):911–915. doi: 10.1128/aem.58.3.911-915.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Resnick S. M., Torok D. S., Gibson D. T. Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase. FEMS Microbiol Lett. 1993 Nov 1;113(3):297–302. doi: 10.1111/j.1574-6968.1993.tb06530.x. [DOI] [PubMed] [Google Scholar]
  31. Saitou N., Nei M. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987 Jul;4(4):406–425. doi: 10.1093/oxfordjournals.molbev.a040454. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Sato S. I., Nam J. W., Kasuga K., Nojiri H., Yamane H., Omori T. Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10. J Bacteriol. 1997 Aug;179(15):4850–4858. doi: 10.1128/jb.179.15.4850-4858.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Senda T., Sugiyama K., Narita H., Yamamoto T., Kimbara K., Fukuda M., Sato M., Yano K., Mitsui Y. Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102. J Mol Biol. 1996 Feb 9;255(5):735–752. doi: 10.1006/jmbi.1996.0060. [DOI] [PubMed] [Google Scholar]
  35. Shine J., Dalgarno L. Determinant of cistron specificity in bacterial ribosomes. Nature. 1975 Mar 6;254(5495):34–38. doi: 10.1038/254034a0. [DOI] [PubMed] [Google Scholar]
  36. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  37. Spence E. L., Kawamukai M., Sanvoisin J., Braven H., Bugg T. D. Catechol dioxygenases from Escherichia coli (MhpB) and Alcaligenes eutrophus (MpcI): sequence analysis and biochemical properties of a third family of extradiol dioxygenases. J Bacteriol. 1996 Sep;178(17):5249–5256. doi: 10.1128/jb.178.17.5249-5256.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Taira K., Hirose J., Hayashida S., Furukawa K. Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707. J Biol Chem. 1992 Mar 5;267(7):4844–4853. [PubMed] [Google Scholar]
  39. Tan H. M., Tang H. Y., Joannou C. L., Abdel-Wahab N. H., Mason J. R. The Pseudomonas putida ML2 plasmid-encoded genes for benzene dioxygenase are unusual in codon usage and low in G+C content. Gene. 1993 Aug 16;130(1):33–39. doi: 10.1016/0378-1119(93)90343-2. [DOI] [PubMed] [Google Scholar]
  40. Thompson J. D., Higgins D. G., Gibson T. J. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res. 1994 Nov 11;22(22):4673–4680. doi: 10.1093/nar/22.22.4673. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  42. Werlen C., Kohler H. P., van der Meer J. R. The broad substrate chlorobenzene dioxygenase and cis-chlorobenzene dihydrodiol dehydrogenase of Pseudomonas sp. strain P51 are linked evolutionarily to the enzymes for benzene and toluene degradation. J Biol Chem. 1996 Feb 23;271(8):4009–4016. doi: 10.1074/jbc.271.8.4009. [DOI] [PubMed] [Google Scholar]
  43. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]
  44. Zabinski R., Münck E., Champion P. M., Wood J. M. Kinetic and Mössbauer studies on the mechanism of protocatechuic acid 4,5-oxygenase. Biochemistry. 1972 Aug 15;11(17):3212–3219. doi: 10.1021/bi00767a012. [DOI] [PubMed] [Google Scholar]
  45. 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]
  46. van der Meer J. R., de Vos W. M., Harayama S., Zehnder A. J. Molecular mechanisms of genetic adaptation to xenobiotic compounds. Microbiol Rev. 1992 Dec;56(4):677–694. doi: 10.1128/mr.56.4.677-694.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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