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. 1996 Oct;178(19):5592–5601. doi: 10.1128/jb.178.19.5592-5601.1996

Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase.

K P Fong 1, C B Goh 1, H M Tan 1
PMCID: PMC178396  PMID: 8824602

Abstract

The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol. Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26. In vitro translation analysis showed bedD to code for a polypeptide of ca. 39 kDa. Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus. A bedD mutant of P. putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth. The bedD gene product was found to complement the todD mutation in P. putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase. The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified. It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol. Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.

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Selected References

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  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Axcell B. C., Geary P. J. Purification and some properties of a soluble benzene-oxidizing system from a strain of Pseudomonas. Biochem J. 1975 Jan;146(1):173–183. doi: 10.1042/bj1460173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Axcell B. C., Geary P. J. The metabolism of benzene by bacteria. Purification and some properties of the enzyme cis-1,2-dihydroxycyclohexa-3,5-diene (nicotinamide adenine dinucleotide) oxidoreductase (cis-benzene glycol dehydrogenase). Biochem J. 1973 Dec;136(4):927–934. doi: 10.1042/bj1360927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. BERTANI G. Studies on lysogenesis. I. The mode of phage liberation by lysogenic Escherichia coli. J Bacteriol. 1951 Sep;62(3):293–300. doi: 10.1128/jb.62.3.293-300.1951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bairoch A. PROSITE: a dictionary of sites and patterns in proteins. Nucleic Acids Res. 1991 Apr 25;19 (Suppl):2241–2245. doi: 10.1093/nar/19.suppl.2241. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Blattner F. R., Burland V., Plunkett G., 3rd, Sofia H. J., Daniels D. L. Analysis of the Escherichia coli genome. IV. DNA sequence of the region from 89.2 to 92.8 minutes. Nucleic Acids Res. 1993 Nov 25;21(23):5408–5417. doi: 10.1093/nar/21.23.5408. [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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  8. Daniel R., Stuertz K., Gottschalk G. Biochemical and molecular characterization of the oxidative branch of glycerol utilization by Citrobacter freundii. J Bacteriol. 1995 Aug;177(15):4392–4401. doi: 10.1128/jb.177.15.4392-4401.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Davison J., Heusterspreute M., Chevalier N., Ha-Thi V., Brunel F. Vectors with restriction site banks. V. pJRD215, a wide-host-range cosmid vector with multiple cloning sites. Gene. 1987;51(2-3):275–280. doi: 10.1016/0378-1119(87)90316-7. [DOI] [PubMed] [Google Scholar]
  10. DiMarco A. A., Ornston L. N. Regulation of p-hydroxybenzoate hydroxylase synthesis by PobR bound to an operator in Acinetobacter calcoaceticus. J Bacteriol. 1994 Jul;176(14):4277–4284. doi: 10.1128/jb.176.14.4277-4284.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. 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]
  12. Ensley B. D., Gibson D. T. Naphthalene dioxygenase: purification and properties of a terminal oxygenase component. J Bacteriol. 1983 Aug;155(2):505–511. doi: 10.1128/jb.155.2.505-511.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Ensor C. M., Tai H. H. Site-directed mutagenesis of the conserved tyrosine 151 of human placental NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase yields a catalytically inactive enzyme. Biochem Biophys Res Commun. 1991 Apr 30;176(2):840–845. doi: 10.1016/s0006-291x(05)80262-1. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Gibson D. T., Hensley M., Yoshioka H., Mabry T. J. Formation of (+)-cis-2,3-dihydroxy-1-methylcyclohexa-4,6-diene from toluene by Pseudomonas putida. Biochemistry. 1970 Mar 31;9(7):1626–1630. doi: 10.1021/bi00809a023. [DOI] [PubMed] [Google Scholar]
  16. Goyal A. K., Zylstra G. J. Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39. Appl Environ Microbiol. 1996 Jan;62(1):230–236. doi: 10.1128/aem.62.1.230-236.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Harayama S., Rekik M., Wasserfallen A., Bairoch A. Evolutionary relationships between catabolic pathways for aromatics: conservation of gene order and nucleotide sequences of catechol oxidation genes of pWW0 and NAH7 plasmids. Mol Gen Genet. 1987 Dec;210(2):241–247. doi: 10.1007/BF00325689. [DOI] [PubMed] [Google Scholar]
  18. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Ismaiel A. A., Zhu C. X., Colby G. D., Chen J. S. Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii. J Bacteriol. 1993 Aug;175(16):5097–5105. doi: 10.1128/jb.175.16.5097-5105.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaniga K., Delor I., Cornelis G. R. A wide-host-range suicide vector for improving reverse genetics in gram-negative bacteria: inactivation of the blaA gene of Yersinia enterocolitica. Gene. 1991 Dec 20;109(1):137–141. doi: 10.1016/0378-1119(91)90599-7. [DOI] [PubMed] [Google Scholar]
  22. Kikuchi Y., Nagata Y., Hinata M., Kimbara K., Fukuda M., Yano K., Takagi M. Identification of the bphA4 gene encoding ferredoxin reductase involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102. J Bacteriol. 1994 Mar;176(6):1689–1694. doi: 10.1128/jb.176.6.1689-1694.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kurkela S., Lehväslaiho H., Palva E. T., Teeri T. H. Cloning, nucleotide sequence and characterization of genes encoding naphthalene dioxygenase of Pseudomonas putida strain NCIB9816. Gene. 1988 Dec 20;73(2):355–362. doi: 10.1016/0378-1119(88)90500-8. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Mallinder P. R., Pritchard A., Moir A. Cloning and characterization of a gene from Bacillus stearothermophilus var. non-diastaticus encoding a glycerol dehydrogenase. Gene. 1992 Jan 2;110(1):9–16. doi: 10.1016/0378-1119(92)90438-u. [DOI] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Mollet B., Iida S., Shepherd J., Arber W. Nucleotide sequence of IS26, a new prokaryotic mobile genetic element. Nucleic Acids Res. 1983 Sep 24;11(18):6319–6330. doi: 10.1093/nar/11.18.6319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Neidle E., Hartnett C., Ornston L. N., Bairoch A., Rekik M., Harayama S. cis-diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short-chain alcohol dehydrogenase superfamily. Eur J Biochem. 1992 Feb 15;204(1):113–120. doi: 10.1111/j.1432-1033.1992.tb16612.x. [DOI] [PubMed] [Google Scholar]
  30. Oesch F., Daly J. Conversion of naphthalene to trans-naphthalene dihydrodiol: evidence for the presence of a coupled aryl monooxygenase-epoxide hydrase system in hepatic microsomes. Biochem Biophys Res Commun. 1972 Feb 25;46(4):1713–1720. doi: 10.1016/0006-291x(72)90807-8. [DOI] [PubMed] [Google Scholar]
  31. Patel T. R., Gibson D. T. Bacterial cis-dihydrodiol dehydrogenases: comparison of physicochemical and immunological protperties. J Bacteriol. 1976 Dec;128(3):842–850. doi: 10.1128/jb.128.3.842-850.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Patel T. R., Gibson D. T. Purification and propeties of (plus)-cis-naphthalene dihydrodiol dehydrogenase of Pseudomonas putida. J Bacteriol. 1974 Sep;119(3):879–888. doi: 10.1128/jb.119.3.879-888.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Persson B., Krook M., Jörnvall H. Characteristics of short-chain alcohol dehydrogenases and related enzymes. Eur J Biochem. 1991 Sep 1;200(2):537–543. doi: 10.1111/j.1432-1033.1991.tb16215.x. [DOI] [PubMed] [Google Scholar]
  34. Reiner A. M. Metabolism of aromatic compounds in bacteria. Purification and properties of the catechol-forming enzyme, 3,5-cyclohexadiene-1,2-diol-1-carboxylic acid (NAD + ) oxidoreductase (decarboxylating). J Biol Chem. 1972 Aug 25;247(16):4960–4965. [PubMed] [Google Scholar]
  35. Rogers J. E., Gibson D. T. Purification and properties of cis-toluene dihydrodiol dehydrogenase from Pseudomonas putida. J Bacteriol. 1977 Jun;130(3):1117–1124. doi: 10.1128/jb.130.3.1117-1124.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. 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]
  38. Simon M. J., Osslund T. D., Saunders R., Ensley B. D., Suggs S., Harcourt A., Suen W. C., Cruden D. L., Gibson D. T., Zylstra G. J. Sequences of genes encoding naphthalene dioxygenase in Pseudomonas putida strains G7 and NCIB 9816-4. Gene. 1993 May 15;127(1):31–37. doi: 10.1016/0378-1119(93)90613-8. [DOI] [PubMed] [Google Scholar]
  39. Subramanian V., Liu T. N., Yeh W. K., Gibson D. T. Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography. Biochem Biophys Res Commun. 1979 Dec 14;91(3):1131–1139. doi: 10.1016/0006-291x(79)91998-3. [DOI] [PubMed] [Google Scholar]
  40. Tan H. M., Cheong C. M. Substitution of the ISP alpha subunit of biphenyl dioxygenase from Pseudomonas results in a modification of the enzyme activity. Biochem Biophys Res Commun. 1994 Oct 28;204(2):912–917. doi: 10.1006/bbrc.1994.2546. [DOI] [PubMed] [Google Scholar]
  41. Tan H. M., Fong K. P. Molecular analysis of the plasmid-borne bed gene cluster from Pseudomonas putida ML2 and cloning of the cis-benzene dihydrodiol dehydrogenase gene. Can J Microbiol. 1993 Apr;39(4):357–362. doi: 10.1139/m93-052. [DOI] [PubMed] [Google Scholar]
  42. Tan H. M., Joannou C. L., Cooper C. E., Butler C. S., Cammack R., Mason J. R. The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2. J Bacteriol. 1994 May;176(9):2507–2512. doi: 10.1128/jb.176.9.2507-2512.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Tan H. M., Mason J. R. Cloning and expression of the plasmid-encoded benzene dioxygenase genes from Pseudomonas putida ML2. FEMS Microbiol Lett. 1990 Nov;60(3):259–264. doi: 10.1016/0378-1097(90)90314-g. [DOI] [PubMed] [Google Scholar]
  44. 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]
  45. Tang C. T., Ruch F. E., Jr, Lin C. C. Purification and properties of a nicotinamide adenine dinucleotide-linked dehydrogenase that serves an Escherichia coli mutant for glycerol catabolism. J Bacteriol. 1979 Oct;140(1):182–187. doi: 10.1128/jb.140.1.182-187.1979. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Vieira J., Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  47. 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]
  48. Yen K. M., Gunsalus I. C. Plasmid gene organization: naphthalene/salicylate oxidation. Proc Natl Acad Sci U S A. 1982 Feb;79(3):874–878. doi: 10.1073/pnas.79.3.874. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Zamanian M., Mason J. R. Benzene dioxygenase in Pseudomonas putida. Subunit composition and immuno-cross-reactivity with other aromatic dioxygenases. Biochem J. 1987 Jun 15;244(3):611–616. doi: 10.1042/bj2440611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. 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]
  51. van der Meer J. R., Zehnder A. J., de Vos W. M. Identification of a novel composite transposable element, Tn5280, carrying chlorobenzene dioxygenase genes of Pseudomonas sp. strain P51. J Bacteriol. 1991 Nov;173(22):7077–7083. doi: 10.1128/jb.173.22.7077-7083.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]

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