Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1997 Apr;179(8):2595–2607. doi: 10.1128/jb.179.8.2595-2607.1997

Molecular characterization of genes of Pseudomonas sp. strain HR199 involved in bioconversion of vanillin to protocatechuate.

H Priefert 1, J Rabenhorst 1, A Steinbüchel 1
PMCID: PMC179009  PMID: 9098058

Abstract

The gene loci vdh, vanA, and vanB, which are involved in the bioconversion of vanillin to protocatechuate by Pseudomonas sp. strain HR199 (DSM 7063), were identified as the structural genes of a novel vanillin dehydrogenase (vdh) and the two subunits of a vanillate demethylase (vanA and vanB), respectively. These genes were localized on an EcoRI fragment (E230), which was cloned from a Pseudomonas sp. strain HR199 genomic library in the cosmid pVK100. The vdh gene was identified on a subfragment (HE35) of E230, and the vanA and vanB genes were localized on a different subfragment (H110) of E230. The nucleotide sequences of fragment HE35 and part of fragment H110 were determined, revealing open reading frames of 1062, 951, and 1446 bp, representing vanA, vanB, and vdh, respectively. The vdh gene was organized in one operon together with a fourth open reading frame (ORF2), of 735 bp, which was located upstream of vdh. The deduced amino acid sequences of vanA and vanB exhibited 78.8 and 62.1% amino acid identity, respectively, to the corresponding gene products from Pseudomonas sp. strain ATCC 19151 (F. Brunel and J. Davison, J. Bacteriol. 170:4924-4930, 1988). The deduced amino acid sequence of the vdh gene exhibited up to 35.3% amino acid identity to aldehyde dehydrogenases from different sources. The deduced amino acid sequence of ORF2 exhibited up to 28.4% amino acid identity to those of enoyl coenzyme A hydratases. Escherichia coli strains harboring fragment E230 cloned in pBluescript SK- converted vanillin to protocatechuate via vanillate, indicating the functional expression of vdh, vanA, and vanB in E. coli. High expression of vdh in E. coli was achieved with HE35 cloned in pBluescript SK-. The resulting recombinant strains converted vanillin to vanillate at a rate of up to 0.3 micromol per min per ml of culture. Transfer of vanA, vanB, and vdh to Alcaligenes eutrophus and to different Pseudomonas strains, which were unable to utilize vanillin or vanillate as carbon sources, respectively, conferred the ability to grow on these substrates to these bacteria.

Full Text

The Full Text of this article is available as a PDF (1.4 MB).

Selected References

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

  1. Aldea M., Claverie-Martín F., Díaz-Torres M. R., Kushner S. R. Transcript mapping using [35S]DNA probes, trichloroacetate solvent and dideoxy sequencing ladders: a rapid method for identification of transcriptional start points. Gene. 1988 May 15;65(1):101–110. doi: 10.1016/0378-1119(88)90421-0. [DOI] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  3. Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
  4. Boyd L. A., Adam L., Pelcher L. E., McHughen A., Hirji R., Selvaraj G. Characterization of an Escherichia coli gene encoding betaine aldehyde dehydrogenase (BADH): structural similarity to mammalian ALDHs and a plant BADH. Gene. 1991 Jul 15;103(1):45–52. doi: 10.1016/0378-1119(91)90389-s. [DOI] [PubMed] [Google Scholar]
  5. Brunel F., Davison J. Cloning and sequencing of Pseudomonas genes encoding vanillate demethylase. J Bacteriol. 1988 Oct;170(10):4924–4930. doi: 10.1128/jb.170.10.4924-4930.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Cartwright N. J., Buswell J. A. The separation of vanillate O-demethylase from protocatechuate 3,4-oxygenase by ultracentrifugation. Biochem J. 1967 Nov;105(2):767–770. doi: 10.1042/bj1050767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cartwright N. J., Smith A. R. Bacterial attack on phenolic ethers: An enzyme system demethylating vanillic acid. Biochem J. 1967 Mar;102(3):826–841. doi: 10.1042/bj1020826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Correll C. C., Batie C. J., Ballou D. P., Ludwig M. L. Phthalate dioxygenase reductase: a modular structure for electron transfer from pyridine nucleotides to [2Fe-2S]. Science. 1992 Dec 4;258(5088):1604–1610. doi: 10.1126/science.1280857. [DOI] [PubMed] [Google Scholar]
  9. Dehmel U., Engesser K. H., Timmis K. N., Dwyer D. F. Cloning, nucleotide sequence, and expression of the gene encoding a novel dioxygenase involved in metabolism of carboxydiphenyl ethers in Pseudomonas pseudoalcaligenes POB310. Arch Microbiol. 1995 Jan;163(1):35–41. doi: 10.1007/BF00262201. [DOI] [PubMed] [Google Scholar]
  10. Denome S. A., Stanley D. C., Olson E. S., Young K. D. Metabolism of dibenzothiophene and naphthalene in Pseudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway. J Bacteriol. 1993 Nov;175(21):6890–6901. doi: 10.1128/jb.175.21.6890-6901.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Farrés J., Guan K. L., Weiner H. Primary structures of rat and bovine liver mitochondrial aldehyde dehydrogenases deduced from cDNA sequences. Eur J Biochem. 1989 Mar 1;180(1):67–74. doi: 10.1111/j.1432-1033.1989.tb14616.x. [DOI] [PubMed] [Google Scholar]
  13. Farrés J., Wang T. T., Cunningham S. J., Weiner H. Investigation of the active site cysteine residue of rat liver mitochondrial aldehyde dehydrogenase by site-directed mutagenesis. Biochemistry. 1995 Feb 28;34(8):2592–2598. doi: 10.1021/bi00008a025. [DOI] [PubMed] [Google Scholar]
  14. Farrés J., Wang X., Takahashi K., Cunningham S. J., Wang T. T., Weiner H. Effects of changing glutamate 487 to lysine in rat and human liver mitochondrial aldehyde dehydrogenase. A model to study human (Oriental type) class 2 aldehyde dehydrogenase. J Biol Chem. 1994 May 13;269(19):13854–13860. [PubMed] [Google Scholar]
  15. Friedrich B., Hogrefe C., Schlegel H. G. Naturally occurring genetic transfer of hydrogen-oxidizing ability between strains of Alcaligenes eutrophus. J Bacteriol. 1981 Jul;147(1):198–205. doi: 10.1128/jb.147.1.198-205.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. Harayama S., Rekik M., Bairoch A., Neidle E. L., Ornston L. N. Potential DNA slippage structures acquired during evolutionary divergence of Acinetobacter calcoaceticus chromosomal benABC and Pseudomonas putida TOL pWW0 plasmid xylXYZ, genes encoding benzoate dioxygenases. J Bacteriol. 1991 Dec;173(23):7540–7548. doi: 10.1128/jb.173.23.7540-7548.1991. [DOI] [PMC free article] [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. Hempel J., Kaiser R., Jörnvall H. Mitochondrial aldehyde dehydrogenase from human liver. Primary structure, differences in relation to the cytosolic enzyme, and functional correlations. Eur J Biochem. 1985 Nov 15;153(1):13–28. doi: 10.1111/j.1432-1033.1985.tb09260.x. [DOI] [PubMed] [Google Scholar]
  20. Inoue J., Shaw J. P., Rekik M., Harayama S. Overlapping substrate specificities of benzaldehyde dehydrogenase (the xylC gene product) and 2-hydroxymuconic semialdehyde dehydrogenase (the xylG gene product) encoded by TOL plasmid pWW0 of Pseudomonas putida. J Bacteriol. 1995 Mar;177(5):1196–1201. doi: 10.1128/jb.177.5.1196-1201.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Knauf V. C., Nester E. W. Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid. Plasmid. 1982 Jul;8(1):45–54. doi: 10.1016/0147-619x(82)90040-3. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Mason J. R., Cammack R. The electron-transport proteins of hydroxylating bacterial dioxygenases. Annu Rev Microbiol. 1992;46:277–305. doi: 10.1146/annurev.mi.46.100192.001425. [DOI] [PubMed] [Google Scholar]
  24. Nakatsu C. H., Straus N. A., Wyndham R. C. The nucleotide sequence of the Tn5271 3-chlorobenzoate 3,4-dioxygenase genes (cbaAB) unites the class IA oxygenases in a single lineage. Microbiology. 1995 Feb;141(Pt 2):485–495. doi: 10.1099/13500872-141-2-485. [DOI] [PubMed] [Google Scholar]
  25. Neidle E. L., Hartnett C., Ornston L. N., Bairoch A., Rekik M., Harayama S. Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases. J Bacteriol. 1991 Sep;173(17):5385–5395. doi: 10.1128/jb.173.17.5385-5395.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Niegemann E., Schulz A., Bartsch K. Molecular organization of the Escherichia coli gab cluster: nucleotide sequence of the structural genes gabD and gabP and expression of the GABA permease gene. Arch Microbiol. 1993;160(6):454–460. doi: 10.1007/BF00245306. [DOI] [PubMed] [Google Scholar]
  27. Nies D., Mergeay M., Friedrich B., Schlegel H. G. Cloning of plasmid genes encoding resistance to cadmium, zinc, and cobalt in Alcaligenes eutrophus CH34. J Bacteriol. 1987 Oct;169(10):4865–4868. doi: 10.1128/jb.169.10.4865-4868.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Otaka E., Ooi T. Examination of protein sequence homologies: V. New perspectives on evolution between bacterial and chloroplast-type ferredoxins inferred from sequence evidence. J Mol Evol. 1989 Sep;29(3):246–254. doi: 10.1007/BF02100208. [DOI] [PubMed] [Google Scholar]
  29. Priefert H., Hein S., Krüger N., Zeh K., Schmidt B., Steinbüchel A. Identification and molecular characterization of the Alcaligenes eutrophus H16 aco operon genes involved in acetoin catabolism. J Bacteriol. 1991 Jul;173(13):4056–4071. doi: 10.1128/jb.173.13.4056-4071.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Priefert H., Krüger N., Jendrossek D., Schmidt B., Steinbüchel A. Identification and molecular characterization of the gene coding for acetaldehyde dehydrogenase II (acoD) of Alcaligenes eutrophus. J Bacteriol. 1992 Feb;174(3):899–907. doi: 10.1128/jb.174.3.899-907.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Pries A., Priefert H., Krüger N., Steinbüchel A. Identification and characterization of two Alcaligenes eutrophus gene loci relevant to the poly(beta-hydroxybutyric acid)-leaky phenotype which exhibit homology to ptsH and ptsI of Escherichia coli. J Bacteriol. 1991 Sep;173(18):5843–5853. doi: 10.1128/jb.173.18.5843-5853.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ribbons D. W. Requirement of two protein fractions for O-demethylase activity in Pseudomonas testosteroni. FEBS Lett. 1971 Jan 12;12(3):161–165. doi: 10.1016/0014-5793(71)80058-3. [DOI] [PubMed] [Google Scholar]
  33. Ribbons D. W. Stoicheiometry of O-demethylase activity in Pseudomonas aeruginosa. FEBS Lett. 1970 May 25;8(2):101–104. doi: 10.1016/0014-5793(70)80235-6. [DOI] [PubMed] [Google Scholar]
  34. SCHLEGEL H. G., KALTWASSER H., GOTTSCHALK G. [A submersion method for culture of hydrogen-oxidizing bacteria: growth physiological studies]. Arch Mikrobiol. 1961;38:209–222. [PubMed] [Google Scholar]
  35. 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]
  36. Stegemann H., Francksen H., Macko V. Potato proteins: genetic and physiological changes, evaluated by one- and two-dimensional PAA-gel-techniques. Z Naturforsch C. 1973 Nov-Dec;28(11):722–732. doi: 10.1515/znc-1973-11-1213. [DOI] [PubMed] [Google Scholar]
  37. Strauss E. C., Kobori J. A., Siu G., Hood L. E. Specific-primer-directed DNA sequencing. Anal Biochem. 1986 Apr;154(1):353–360. doi: 10.1016/0003-2697(86)90536-1. [DOI] [PubMed] [Google Scholar]
  38. Tinoco I., Jr, Borer P. N., Dengler B., Levin M. D., Uhlenbeck O. C., Crothers D. M., Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. doi: 10.1038/newbio246040a0. [DOI] [PubMed] [Google Scholar]
  39. Toms A., Wood J. M. The degradation of trans-ferulic acid by Pseudomonas acidovorans. Biochemistry. 1970 Jan 20;9(2):337–343. doi: 10.1021/bi00804a021. [DOI] [PubMed] [Google Scholar]
  40. Walker J. E., Fearnley I. M., Gay N. J., Gibson B. W., Northrop F. D., Powell S. J., Runswick M. J., Saraste M., Tybulewicz V. L. Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria. J Mol Biol. 1985 Aug 20;184(4):677–701. doi: 10.1016/0022-2836(85)90313-4. [DOI] [PubMed] [Google Scholar]
  41. Wang X., Weiner H. Involvement of glutamate 268 in the active site of human liver mitochondrial (class 2) aldehyde dehydrogenase as probed by site-directed mutagenesis. Biochemistry. 1995 Jan 10;34(1):237–243. doi: 10.1021/bi00001a028. [DOI] [PubMed] [Google Scholar]
  42. Xu J., Johnson R. C. aldB, an RpoS-dependent gene in Escherichia coli encoding an aldehyde dehydrogenase that is repressed by Fis and activated by Crp. J Bacteriol. 1995 Jun;177(11):3166–3175. doi: 10.1128/jb.177.11.3166-3175.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Yamaguchi M., Fujisawa H. Subunit structure of oxygenase component in benzoate-1,2-dioxygenase system from Pseudomonas arvilla C-1. J Biol Chem. 1982 Nov 10;257(21):12497–12502. [PubMed] [Google Scholar]
  44. Yoshida A., Huang I. Y., Ikawa M. Molecular abnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals. Proc Natl Acad Sci U S A. 1984 Jan;81(1):258–261. doi: 10.1073/pnas.81.1.258. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Zheng C. F., Weiner H. Role of the highly conserved histidine residues in rat liver mitochondrial aldehyde dehydrogenase as studied by site-directed mutagenesis. Arch Biochem Biophys. 1993 Sep;305(2):460–466. doi: 10.1006/abbi.1993.1447. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES