Microbiology and Biotechnology Letters (한국미생물·생명공학회지)

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
권호 표지

Cloning Genes Involved in Aniline Degradation from Delftia acidovorans.

Delftia acidovorans로부터 Aniline 분해관련 유전자의 분리

  • 김현주 (한국화학연구원 생물화학부) ;
  • 김성은 (충남대학교 농생물학과) ;
  • 김정건 (서울대학교 응용생물화학부) ;
  • 김진철 (한국화학연구원 생물화학부) ;
  • 최경자 (한국화학연구원 생물화학부) ;
  • 김흥태 (충북대학교 농생물학과) ;
  • 황인규 (서울대학교 응용생물화학부) ;
  • 김홍기 (충남대학교 농생물학과) ;
  • 조광연 (한국화학연구원 생물화학부)
  • Published : 2003.03.01
Download PDF
⟨ Previous Next ⟩

Abstract

Delftia acidovorans 51-A isolated from river water degrades aniline. In order to clone genes involved in aniline degradation, transposon Tn5-B20 was inserted into the strain 51-A to generate a mutant strain 10-4-2 that cannot utilize aniline as a carbon source. The mutant strain was not an auxotroph but could not degrade aniline. Southern hybridization analysis indicated that the transposon was inserted into the mutant bacterial DNA as a single copy. Flanking DNA fragment of Tn5-B2O insertion was cloned and sequenced. DNA sequence analysis revealed three ORFs encoding TdnQ, TdnT, and TdnA 1 that arc responsible for catechol formation from aniline through oxidative deamination. The analysis also confirmed that Tn5-B2O was inserted at the immediate downstream of tdnA1. The result suggests that the transposon insertion behind tdirA1 disrupted the pathway of the catechol formation from aniline, resulting in the mutant phenotype, which cannot degrade aniline. A large plasmid over 100-kb in size was detected from D. acidovorans 51-A and Southern hybridization analysis with Tn5-B2O probe showed that the transposon was inserted on the plasmid named pTDN51. Our results indicated that the tdn genes on pTDN51 of D. acidovorans 51-A are involved in aniline degradation.

아닐린을 분해할 수 있는 Deiftia acidovoran 51-A가 기존에 분리되어 아닐린 분해능이 우수함이 보고되었다. 이 균주로부터 아닐린 분해관련 유전자를 선발하기 위하여 Tn5-B20삽입 변이체들을 유도하여 영양요구형이 아니면서 아닐린을 분해할 수 없는 변이균주 D. acidovorans 10-4-2 균주를 선발하였다. Southern hybridization 결과 이 변이균주에는 Tn5-B20이 한 copy만 삽입된 것으로 나타났다 이 변이균주의 Tn5-B20삽입의 인접 유전자들을 분리하여 염기서열을 분석한 결과 아닐린 분해의 첫 단계에 해당하는 아닐린의 catechol로의 산화적 deamination에 관련되어 있는 것으로 추정하는 tdnQ, tdnT tdnAl 유전자들이 동정되었고 Tn5-B2O은 tdnAl의 바로 밑에 삽입된 것을 알 수 있었다. TdnA2 및 downstream의 유전자 기능을 상실하여 아닐린을 catechol로 전환하는 과정에 변이가 발생하고 따라서 아닐린을 탄소원으로 이용하지 못하는 표현형을 가지게 된 것으로 결론지을 수 있었다. Tn5-B20 의 일부 DNA 조각을 probe로 Southern hybridization 결과 transposon 삽입이 대형의 플라스미드에 삽입된 것으로 나타나 tdn 유전자들이 pTDN51이라고 명명한 100-kb 이상의 대형 플라스미드에 위치함을 알 수 있었다. 이상의 결과는 D. acidovorans 51-A의 pTDN51상의 tdn유전자들이 아닐린의 분해에 관여함을 보여준다.

Keywords

References

  1. Agric. Biol. Chem. v.48 Rapid biodegradation of aniline by Frateuria species ANA-18 and its aniline metabolism Aoki, K.;K. Ohtsuka;R. Shinke;H. Nishima. https://doi.org/10.1271/bbb1961.48.865
  2. J. Agric. Food Chem. v.26 Adsorption and transformation of four substituted anilines in soil Bollag,J.M.;P.Blattmann;T.Laanio https://doi.org/10.1021/jf60220a011
  3. Appl. Environ. Microbiol v.67 Genetic diversity among 3-chloroaniline- and aniline-degrading strains of the Comamonadaceae Boon,N.;J.Goris;P.De Vos;W.Verstraete;E.M.Top https://doi.org/10.1128/AEM.67.3.1107-1115.2001
  4. Food. Chem. Toxicol. v.28 Toxicity of p-chloroaniline in rats and mice Chabra, R. S.;M.Thompson;M. R. Elwell;A. C. Peters. https://doi.org/10.1016/0278-6915(90)90148-G
  5. Microbiology v.143 Plasmid-encoded genes specifying aniline oxidation from Acinetobacter sp. strain YAA Fujii, T.;M. Takeo;Y. Maeda. https://doi.org/10.1099/00221287-143-1-93
  6. J. Bacteriol. v.179 Nucleotide sequences and regulational analysis of genes involved in conversion of aniline to catechol in Pseudomonas putida UCC22(pTDNI) Fukumori,F.;C.P.Saint.
  7. Water Sci. Technol v.36 Biodegradation of aniline Gheewala,S.H.;A.P.Annachhatre
  8. FEMS Microbiol. Rev. v.103 Microbial breakdown o fhalogenated aromatic pesticides an related compounds Haggblom,M.M. https://doi.org/10.1111/j.1574-6968.1992.tb05823.x
  9. J. Bacteriol v.145 Rapid procedure for detection and isolation of large and small plasmids Kado,C.I.;S.T.Liu
  10. Herbicides: chemistry, degradation, and mode of action(2nd ed.) Keamey,P.C.;D.D.Kaufman
  11. Gene v.70 Improved broad-host-range plasmids for DNA cloning in gram-negative bacteria Keen,N.T.;S.Tamaki;D.Kobayashi;D.Trollinger https://doi.org/10.1016/0378-1119(88)90117-5
  12. Kor, J. Appl. Microbiol. Biotechnol. v.28 Isolation and characterization of Stenotrophomonas maltophilia strains capable of degrading aniline Kim, H.-J.;J.-C. Kim;H. T. Kim;G. J. Choi;D. Choi;H. G. Kim;K. Y. Cho.
  13. FEMS Microbiol. Lett. v.111 Isolation and characterization of a subsurface bacterium that degrades aniline and methylanilines Konopka,A. https://doi.org/10.1111/j.1574-6968.1993.tb06367.x
  14. Arch. Microbiol. v.155 Degradation of aniline and monochlorinated anilines by soil-born Pseudomonas acidovorans strains Loidl, M.;c. Hinteregger;G. Ditzelmuller;A. Ferschl;F. Streichsbier https://doi.org/10.1007/BF00291275
  15. Appl. Environ. Microbiol v.48 Mechanisms and pathway of aniline elimination from aquatic environments Lyons,C.D.;S.Katz;R.Bartha
  16. J. Gen. Microbiol. v.133 pTDN1, a catabolic plasmid involved in aromatic amine catabolism in Pseudomonas putida mt-2 McClure, N. C.;W. A. Venables.
  17. Gene v.133 Construction and use of a new broad-host-range lacZ transcriptional fusion vector Parales,R.E.;C.S.Harwood https://doi.org/10.1016/0378-1119(93)90220-W
  18. Biochem. Biophys. Res. Commun v.15 Isolation and properties of an iron-protein from the (reduced coenzyme Q )-cytochrome C reductase complex of the respiratory chain Rieske,J.S.;D.H.MacLennan;R.Coleman https://doi.org/10.1016/0006-291X(64)90171-8
  19. Molecular cloning: a laboratory manual(2nd ed.) Sambrook,J.;E.F.Fritsch;T.Maniatis
  20. Arch. Microbiol v.153 Anaerobic aniline degradation via reductive deamination of 4-aminobenzoyl-CoA in Desulfobacterium anilini Schneel,S.;B.Schink
  21. Gene v.81 New derivatices of transposon Tn5 suitable for mobilization of replicons generation of operon fusions and induction of genes in Gramnegative bacteria Simon, R.;J. Quandt;W. Klipp.
  22. Bio/Technology v.1 A broad host range mobilization sysem for in vivo genetic engineering: transposon mutagenesis in Gram negative bacteria Simon,R.;U.Priefer;A.Puhler https://doi.org/10.1038/nbt1183-784
  23. Carcinogenesis v.17 Neoplastic transformation and DNA-binding of 2-chloroaniline in SV40-immortalized human uroepithelial cell lines Swaminathan, S.;S. M. Frederickson;J. F. Hatcher;C. A. Reznikoff;M. A. Butler;K. L. Cheever;R. E. Savage https://doi.org/10.1093/carcin/17.4.857
  24. strain YAA. J. Ferment. Bioeng v.85 Sequence analysis of the genes encoding a multicomponent dioxygenase involved in oxidation of aniline and o-toluidine in Acinetobacter sp. Takeo,M.;T.Fujii;Y.Maeda https://doi.org/10.1016/S0922-338X(97)80347-9
  25. Gene v.33 Improved M13 phage cloning vectors and host strains: nucleotide sequences fo the M13mp18 and pUC vectors Yanisch-Perron, C.;J. Vieira;J. Messing. https://doi.org/10.1016/0378-1119(85)90120-9
  26. Methods Microbiol v.17 Conjugation Willets,N. https://doi.org/10.1016/S0580-9517(09)70051-0
  27. Appl. Environ. Microbiol. v.50 Microbial mineralization of ring-substituted aniliness through an ortho-cleavage pathway Zeyer, J.;A. Wasserfallen;K. N. Timmis.
  28. Appl. Environ. Microbiol v.65 The alkene monooxygenase from Xanthobacter strain Py2 is closely related to aromatic monooxygenases an catalyzes aromatic monohydroxylation of benzene, toluene, and phenol Zhou,N.Y.;A.Jenkins;C.K.N.Chan Kwo Chion;D.J.Leak