Korean Journal of Microbiology (미생물학회지)

The Microbiological Society of Korea (한국미생물학회)
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Structure Analysis of pmcABCDEFT Gene Cluster for Degradation of Protocatechuate from Comamonas sp. Strain DJ-12

Comamonas sp. Strain DJ-12로부터 Protocatechuate의 분해에 관여하는 pmcABCDEFT 유전자군의 구조 분석

  • Kang Cheol-Hee (Department of Microbiology. Chongbuk National University) ;
  • Lee Sang-Mhan (Department of Life Science, Cheongju University) ;
  • Lee Kyoung (Department of Microbiology. Changwon National University) ;
  • Lee Dong-Hun (Department of Microbiology. Chongbuk National University) ;
  • Kim Chi-Kyung (Department of Microbiology. Chongbuk National University)
  • 강철희 (충북대 자연대 미생물학과) ;
  • 이상만 (청주대학교 이공대 생명과학과) ;
  • 이경 (창원대학교 자연대 미생물학과) ;
  • 이동훈 (충북대 자연대 미생물학과) ;
  • 김치경 (충북대 자연대 미생물학과)
  • Published : 2005.09.01
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Abstract

Comamonas sp. strain DJ-12 is a bacterial isolate capable of degrading of 4-chlorobiphenyl (4CB) as a carbon and energy source. The degradation pathway was characterized as being conducted by consecutive reactions of the meta-degradation of 4CB, hydrolytic dechlorination of 4-chlorobenzoate (4CBA), hydroxylation of 4-hydroxybenzoate, and meta-degradation of protocatechuate to product TCA metabolites. The 6.8 kb fragment from the chromosomal DNA of Comamonas sp. strain DJ-12 included the genes encoding for the meta-degradation of PCA; the genes of protocatechuate 4,5-dioxygenase alpha and beta subunits (pmcA and pmcB), 4-carboxy-2-hydroxymuconate-6-semialdehyde dehydrogenase (pmcC), 2-pyrone-4,6-dicarboxylate hydrolase (pmcD), 4-oxalomesaconate (OMA) hydratase(pmcE), 4-oxalocitramalate (OCM) aldolase (pmcF), and transporter gene (pmcT). They were organized in the order of pmcT-pmcE-pmcF-pmcD-pmcA-pmcB-pmcC. The amino acid sequences deduced from the nucleotide sequences of pmcABCDEFT genes from Comamonas sp. strain DJ-12 exhibited 94 to $98\%$ homologies with those of Comamonas testosteroni BR6020 and Pseudomonas ochraceae NGJ1, but only 52 to $74\%$ with homologies Sphingomonas paucimobilis SYK-6, Sphingomonas sp. LB126, and Arthrobacter keyseri 12B.

Comamonas sp. strain DJ-12의 pmcABCDEFT 유전자군은 protocatechuate (PCA)의 분해과정에 관여하는 PCA 4,5-dioxygenase, 4-carboxy-2hydroxymuconic semialdehyde (CHMS) dehydrogenase, 2-pyrone04,5-dicarboxylate(PDC) hydrolase, 4-oxalomesaconate (OMA) hydratase, 그리고 4-oxalocitramalate (OCM) aldolase 등의 효소들을 생산하는 유전자들과 transporter의 역학을 하는 유전자로 각각 확인되었다. 이 유전자군은 Comamonas sp. strain DJ-12의 chromosomal DNA로부터 얻은 PCR 산물들을 T-vector에 ligation하여 재조합 플라스미드 pMT1, pMT2, pMT3, pMT4, pMT5, pMT6, pMT7, pMT8, pMT9, pMT10을 제조하였다. 이들 재조합 플라스미드의 염기서열을 분석한 결과 PCA 4,5-dioxygenase 유전자는 alpha(pmcA)와 beta(pmcB) 두 개의 subunit으로 구성 되어있으며, 각각 450 bp와 870 bp이었다. CHMS dehydrogenase 유전자(pmcC)는 960 bp, PDC hydrolase 유전자(pmcD)는 918 bp이였으며, OMA hydratase 유전자(pmcE)는 1029 bp, OCM aldolase 유전자 (pmcF)는 689 bp, 그리고 transporter 유전자(pmcT)는 1,398 bp이였다. 이들 pmc 유전자들은 pmcT-pmcE-pmcF-pmcD-pmcA-pmcB-pmcC의 순서로 배열되어 있었다. Comamonas sp. strain DJ-12의 pmcABCDEFT 유전자산물의 아미노산 서열을 분석한 결과, Comamonas testosteroni BR6020 및 Psedomonas ochraceae NG.J1와 $94{\~}98\%$의 높은 유사성을 보였고, 그 유전자들의 배열 순서도 동일하였다. 그러나 Sphingomonas paucimobilis SYK-6, Sphingomonas sp. LB126, 그리고 Arthrobacter keyser 12B와는 아미노산 서열이 $52{\~}74\%$의 유사성을 보였고, 그 유전자의 배열 구조도 상이하였다.

Keywords

References

  1. Arciero, D.M., A.M. Orville, and J.D. Lipscomb. 1990. Protocatechuate 4,5-dioxygenase from Pseudomonas testosteroni. Methods Enzymol. 188, 89-95 https://doi.org/10.1016/0076-6879(90)88017-5
  2. Chae, J.C., E.H. Kim, S.H. Park, and C.K. Kim. 2000. Catabolic degradation of 4-chlorobiphenyl by Pseudomonas sp. DJ-12 via consecutive reaction of meta-cleavage and hydrolytic dechlorination. Biotechnol. Bioprocess Eng. 5, 449-455 https://doi.org/10.1007/BF02931946
  3. Chae, J.C., Y. Kim, Y.C. Kim, G.J. Zylstra, and C.K. Kim. 2000. Genetic structure and functional implication of the fcb gene cluster for hydrolytic dechlorination of 4-chlorobenzoate from Pseudomonas sp. DJ-12. Gene 258, 109-116 https://doi.org/10.1016/S0378-1119(00)00419-4
  4. Eaton, R.W. 2001. Plasmid-encoded phthalate catabolic pathway in Arthrobacter keyseri 12B. J. Bacteriol. 183, 3689-3703 https://doi.org/10.1128/JB.183.12.3689-3703.2001
  5. Goyal, A.K. and G.J. Zylstra. 1996. Molecular cloning of novel genes for polycyclic aromatic hydrocarbon degradation from Comamonas testosteroni GZ39. Appl. Environ. Microbiol. 62, 230-236
  6. Karegoudar, T.B., J.C. Chae, and C.K. Kim. 1999. Catabolism of 4-hydroxy benzoic acid by Pseudomonas sp. DJ-12. J. Microbiol. 37, 123-127
  7. Katayama, Y., S. Nishikawa, M. Nakamura, K. Yano, M. Yamasaki, N. Morohoshi, and T. Haraguchi. 1987. Cloning and expression of Pseudomonas paucimobilis SYK-6 genes involved in the degradation of vanillate and protocatechuate in P. putida. Mokuzai Gakkaisi 33, 77-79
  8. Kim, J.W., C.K. Kim, Y.C. Kim, J.H. Yeoum, and J.G. Lee. 1987. Isolation and characterization of bacteria degrading chlorinated aromatic hydrocarbons. J. Microbiol. 25, 122-128
  9. Lee, J.H., D.W. Park, C.H. Kang, J.C. Chae, D.H. Lee, and C.K. Kim. 2004. Reidentification of Comamonas sp. strain DJ-12 and analysis of its pcbABC2D2 genes responsible for degradation of 4-chlorobiphenyl. Kor. J. Microbiol. 40, 121-126
  10. Locher, H.H., T. Leisinger, and A.M. Cook. 1991. 4-sulphobenzoate 3,4-dioxygenase: purification and properties of a desulphonative two-component enzyme system from Comamonas testosteroni T-2. J. Biochem. 274, 833-842 https://doi.org/10.1042/bj2740833
  11. Locher, H.H., C. Malli, S. Hooper, T. Vorherr, T. Leisinger, and A. M. Cook. 1991. Degradation of p-toluic acid (p-toluenecarboxylic acid) and p-toluenesulphonic acid via oxygenation of the methyl sidechain is initiated by the same set of enzymes in Comamonas testosteroni T-2. J. Gen. Microbiol. 137, 220-228
  12. Maruyama, K. 1990. Purification and properties of 4-hydroxy-4-methyl- 2-oxoglutarate aldolase from Pseudomonas ochraceaegrown on phthalate. J. Biochem. 108, 327-333 https://doi.org/10.1093/oxfordjournals.jbchem.a123201
  13. Maruyama, K., T. Shibayama, A. Ichikawa, Y. Sakou, S. Yamada, and H. Sugisaki. 2004. Cloning and characterization of the genes encoding enzymes for the protocatechuate meta-degradation pathway of Pseudomonas ochraceae NGJ1. Biosci. Biotechnol. Biochem. 68, 1434-1441 https://doi.org/10.1271/bbb.68.1434
  14. Masai, E., S. Shinohara, H. Hara, S. Nishikawa, Y. Katayama, and M. Fukuda. 1999. Genetic and biochemical characterization of a 2-pyrone-4,6-dicarboxylic acid hydrolase involved in the protocatechuate 4,5-cleavage pathway of Sphingomonas paucimobilis SYK-6. J. Bacteriol. 181, 55-62
  15. Masai, E., K. Momose, H. Hara, S. Nishikawa, Y. Katayama, and M. Fukuda. 2000. Genetic and biochemical characterization of 4-carboxy-2-hydroxy-muconate-6-semialdehyde dehydrogenase and its role in the protocatechuate 4,5-cleavage pathway in Sphingomonas paucimobilis SYK-6. J. Bacteriol. 182, 6651-6658 https://doi.org/10.1128/JB.182.23.6651-6658.2000
  16. Noda, Y., S. Nishikawa, K. Shiozuka, H. Kadokura, H. Nakajima, K. Yoda, Y. Katayama, N. Morohoshi, T. Haraguchi, and M. Yamasaki. 1990. Molecular cloning of the protocatechuate 4,5-dioxygenase genes of Pseudomonas paucimobilis. J. Bacteriol. 172, 2704-2709 https://doi.org/10.1128/jb.172.5.2704-2709.1990
  17. Alison, B., L.S. Collier, E.L. Neidle, and M.A. Moran. 2000. Key aromatic-ring-cleaving enzyme, protocatechuate 3,4-dioxygenase, in the ecologically important marine Roseobacter lineage. Appl. Environ. Microbiol. 66, 4662-4672 https://doi.org/10.1128/AEM.66.11.4662-4672.2000
  18. Providenti, M.A., J. Mampel, S. MacSween, A.M. Cook, and R.C. Wyndham. 2001. Comamonas testosteroni BR6020 possesses a single genetic locus for extradiol cleavage of protocatechuate. Microbiology 147, 2157-2167 https://doi.org/10.1099/00221287-147-8-2157
  19. Sambrook, J., E.F. Fritsch, and T. Maniatis. 1989. Molecular coling, A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York
  20. Schlfli, H.R., M.A. Weiss, T, Leisinger, and A.M. Cook. 1994. Terephthalate 1,2-dioxygenase system from Comamonas testosteroni T-2: purification and some properties of the oxygenase component. J. Bacteriol. 176, 6644-6652 https://doi.org/10.1128/jb.176.21.6644-6652.1994
  21. Thompson, J.D., D.G. Higgins, and T.J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl. Acids Res. 22, 4673-4680 https://doi.org/10.1093/nar/22.22.4673
  22. Thompson, J.D., T.J. Gibson, F. Plewniak, F. Jeanmougin, and D. G. Higgins. 1997. The Clustal X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucl. Acids Res. 24, 4876-4882
  23. Yun, S.H., C.Y. Yun, and S.I. Kim. 2004. Characterization of protocatechuate 4,5-dioxygenase induced from p-hydroxybenzoate-cultured Pseudomonas sp. K82. J. Microbiol. 42, 152-155
  24. Wattiau, P., L. Bastiaens, R. van Herwijnen, L. Dal, J. R. Parsons, M.E. Renard, D. Springael, and G.R. Cornelis. 2001. Fluorene degradation by Sphingomonas sp. LB126 proceeds through protocatechuic acid: a genetic analysis. Res. Microbiol. 152, 861-872 https://doi.org/10.1016/S0923-2508(01)01269-4
  25. Wolgel, S.A., J.E. Dege, P.E. Perkins-Olson, C.H. Jaurez-Garcia, R.L. Crawford, E. Munck, and J.D. Lipscomb. 1993. Purification and characterization of protocatechuate 2,3-dioxygenase from Bacillus macerans: a new extradiol catecholic dioxygenase. J. Bacteriol. 175, 4414-4426 https://doi.org/10.1128/jb.175.14.4414-4426.1993