Versatile Catabolic Properties of Tn4371-encoded bph Pathway in Comamonas testosteroni (Formerly Pseudomonas sp.) NCIMB 10643

  • Kim, Jong-Soo (Department of Microbiology and Institute of Genetic Engineering, Changwon National University) ;
  • Kim, Ji-Hyun (Department of Microbiology and Institute of Genetic Engineering, Changwon National University) ;
  • Ryu, Eun-Kyeong (Department of Microbiology and Institute of Genetic Engineering, Changwon National University) ;
  • Kim, Jin-Kyoo (Department of Microbiology and Institute of Genetic Engineering, Changwon National University) ;
  • Kim, Chi-Kyung (Department of Microbiology, Chungbuk National University) ;
  • Hwang, In-Gyu (School of Agricultural Biotechnology, Seoul National University) ;
  • Lee, Kyoung (Department of Microbiology and Institute of Genetic Engineering, Changwon National University)
  • Published : 2004.04.01


Comamonas testosteroni (formerly Pseudomonas sp.) NCIMB 10643 can grow on biphenyl and alkylbenzenes $(C_2-C_7)$ via 3-substituted catechols. Thus, to identify the genes encoding the degradation, transposon-mutagenesis was carried out using pAG408, a promoter-probe mini-transposon with a green fluorescent protein (GFP), as a reporter. A mutant, NT-1, which was unable to grow on alkylbenzenes and biphenyl, accumulated catechols and exhibited an enhanced expression of GFP upon exposure to these substrates, indicating that the gfp had been inserted in a gene encoding a broad substrate range catechol 2,3-dioxygenase. The genes (2,826 bp) flanking the gfp cloned from an SphI-digested fragment contained three complete open reading frames that were designated bphCDorfl. The deduced amino acid sequences of bphCDorfl were identical to 2,3-dihydroxybiphenyl 1,2-dioxygenase (BphC), 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (BphD), and OrfI, respectively, that are all involved in the degradation of biphenyl/4-chlorobiphenyl (bph) by Ralstonia oxalatica A5. The deduced amino acid sequence of the orfl revealed a similarity to those of outer membrane proteins belonging to the OmpW family. The introduction of the bphCDorfl genes enabled the NT-l mutant to grow on aromatic hydrocarbons. In addition, PCR analysis indicated that the DNA sequence and gene organization of the bph operon were closely related to those in the bph operon from Tn4371 identified in strain A5. Furthermore, strain A5 was also able to grow on a similar set of alkylbenzenes as strain NCIMB 10643, demonstrating that, among the identified aromatic hydrocarbon degradation pathways, the bph degradation pathway related to Tn4371 was the most versatile in catabolizing a variety of aromatic hydrocarbons of mono- and bicyclic benzenes.



  1. Nucleic Acids Res. v.25 Gapped BLAST and PSI-BLAST: A new generation of protein database search programs Altschul,S.F.;T.L.Madden;A.A.Schaffer;J.Zhang;Z.Zang;W.Miller;D.J.Lipmen
  2. J. Struct. Biol. v.131 Expression, two-dimensional crystallization, and three-dimensional reconstruction of the β-8 outer membrane protein Omp21 from Comamonas acidovorans Baldermann,C.;H.Engelhardt
  3. J. Bacteriol. v.180 The regulated outer membrane protein Omp21 from Comamonas acidovorans is identified as a member of a new family of eight-stranded beta-sheet proteins by its sequence and properties Baldermann,C.;A.Lupas;J.Lubieniecki;H.Engelhardt
  4. J. Ind. Microbiol. Biotechnol. v.25 Toluene degradation pathway from Pseudomonas putida F1: Substrate specificity and gene induction by 1-substituted benzenes Cho,M.C.;D.O.Kang;B.D.Yoon;K.Lee
  5. Microbiology v.149 Expansion of growth substrate range in Pseudomonas putida F1 by mutations in both cymR and todS, which recruit a ring-fission hydrolase CmtE and induce the tod catabolic operon, respetively Choi,E.N.;M.C.Cho;Y.Kim;C.K.Kim;K.Lee
  6. Protein Eng. v.10 Prediction of transmembrane alpha-helices in prokaryotic membrane proteins: The dense alignment surface method Cserzo,M.;E.Wallin;I.Simon;G. von Heijne;A.Elofsson
  7. Mol. Microbiol. v.19 Regulatory noise in prokaryotic promoters: how bacteria learn to respond to novel environmental signals de Lorenzo, V.;J.Perez-Martin
  8. J. Bacteriol. v.175 Metabolism of dibenzothiophene and naphthalene in Psudomonas strains: complete DNA sequence of an upper naphthalene catabolic pathway Denome,S.A.;D.C.Stanley;E.S.Olson;K.D.Young
  9. J. Bacteriol. v.176 Organization and evolution of naphthalene catabolic pathways: Sequence of the DNA encoding 2-hydroxychromene-2-carboxylate isomerase and trans-o-hydroxybenzylidenepyruvate hydratase-aldolase from the NHH7 plasmid Eston,R.W.
  10. Biodegradation v.9 Isopropylbenzene catabolic pathway in Pseudomonase putida RE204: nucleotide sequence analysis of the ipb operon and neighboring DNA from pRE4 Eaton,R.W.;O.V.Selifonova;R.M.Gedney
  11. J. Bacteriol. v.168 Characterization of a plasmid-specified pathway for catabolism of isopropylbenzene in Pseudomonas putida RE204 Eaton,R.W.;K.N.Timmis
  12. J. Bacteriol. v.180 A gene cluster encoding steps in conversion of naphthalene to gentisate in Pseudomonas sp. strain U2 Fuenmayor,S.L.;M.Wild;A.L.Boyes;P.A.Williams
  13. Biochem. Biophys. Res. Commun. v.202 Identification of the bphA and bphB genes of Pseudomonas sp. strains KKS102 involved in degradation of biphenyl and polychlorinated biphenyls Fukuda,M.;Y.Yasukochi;Y.Kikuchi;Y.Nagata;K.Kimbara;H.Horiuchi;M.Takagi;K.Yano
  14. J. Bacteriol. v.175 Gene components responsible for discrete substrate specificity in the metabolism of biphenyl (bph operon) and toluene (tod operon) Furukawa,K.;J.Hirose;A.Suyama;T.Zaili;S.Hayashida
  15. J. Antimicrob. Chemother. v.43 Identification fo oprG, a gene encoding a major outer membrane protein of Pseudomonas aeruginosa Gensberg,K.;A.W.Smith;F.S.Brinkman;R.E.Hancock
  16. Biochemistry v.12 Initial reactions in the oxidation of ethylbenzene by Pseudomonas putida Gibson,D.T.;B.Gschwendt;W.K.Yeh;V.M.Kobal
  17. Curr. Opin. Biotechnol. v.11 Aromatic hydrocarbon dioxygenases in environmental biotechnology Gibson,D.T.;R.E.Parales
  18. Microbial Degradation of Organic Compounds Gibson,D.T.;V.Suramanian
  19. Appl. Environ. Microbiol. v.62 Analysis of cumene (isopropylbenzene) degradation genes from Pseudomonas fluorescens IP01 Habe,H.;K.Kasuga;H.Nojiri;H.Yamane;T.Omori
  20. Appl. Environ. Microbiol. v.68 Flow cytometry analysis of changes in the DNA content of the polychlorinated biphenyl degrader Comamonas testosteroni TK102: Effcet of matabolites on cell-cell separation Hiraoka,Y.;T.Yamada;K.Tone;Y.Futaesaku;K.Kimbara
  21. Gene v.144 THe biphenyl/polychlorinated biphenyl-degradation locus (bph) of Pseudomonas sp. LB400 encodes four additional metabolic enzymes Hofer,B.;S.Backhaus;K.N.Timmis
  22. Nucleic Acids Res. v.18 Nucleotide sequence of the gene, ompW, encodng a 22 kDa immunogenic outer membrane protein of Vibrio cholerae Jalajakumari,M.B.;P.A.Manning
  23. Mothods for General and Molecular Bacteriology Similarity analyses of rRNAs Johnson,J.L.;P.Gerhardt(ed.);R.G.E.Murray(ed.);W.A.Food(ed.);N.R.Krieg(ed.)
  24. J. Bacteriol. v.176 Identification of the bphA4 gene encoding ferredoxin reducates involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102 Kikuchi,Y.;Y.Nagata;M.Hinata;K.Kimbara;M.Fukuda;K.Yano;M.Takagi
  25. J. Bacteriol. v.176 Nucleotide sequence and functional analysis of the meta-cleavage pathway involved in biphenyl and polychlorinated biphenyl degradation in Pseudomonas sp. strain KKS102 Kikuchi,Y.;Y.Yasukochi;Y.Nagata;M.Fukuda;M.Takagi
  26. Appl. Environ. Microbiol. v.68 Monocyclic aromatic hydrocarbon degradation by Rhodococcus sp. strain DK17 Kim,D.;Y.S.Kim;S.K.Kim;S.W.Kim;G.J.Zylstra;Y.M.Kim;E.Kim
  27. Appl. Environ. Microbiol. v.62 Genetic structures of the genes encoding 2,3-dihydroxybiphenyl 1,2-dixoygenase and 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase from biphenyl-and 4-chlorobiphenyl-degrading Pseudomonas sp. strain DJ-12 Kim,E.;Y.Kim;C.K.Kim
  28. Gene v.166 Four new derivatives of the broad-host-range cloning vector pBBR1MCS, carrying different antibiotic-restance cassettes Kovach,M.E.;P.H.Elzer;D.S.Hill;G.T.Robertson;M.A.Farris;R.M.Roop,2nd;K.M.Peterson
  29. Gene v.146 Sequence and expression of the todGIH genes involved in the last three steps of toluene degradation by Pseudomonas putida F1 Lau,P.C.;H.Bergeron;D.Labbe;Y.Wang;R.Borusseau;D.T.Gibson
  30. J. Microbiol. Biotechnol. v.8 Involvement of electrostatic interactions between the components of toluene dioxygenase from Psudomonas putida F1 Lee,K.
  31. Biochem. J. v.118 The microbial metabolism of biphenyl Lung,D.;W.C.Evans
  32. Mol. Gen. Genet. v.253 Organisation of the bph gene cluster of transposon Tn4371, encoding enzymes for the degradation of biphenyl and 4-chlorobiphenyl compounds Merlin,C.;D.Springael;M.Mergeay;A.Toussaint
  33. Mol. Plant Microbe Interact. v.13 Improveed gfp and inaZ broad-host-range promoter-probe vectors Miller,W.G.;J.H.Leveau;S.E.Lindow
  34. J. Bacteriol. v.171 Cloning and expression in Escherichia coli of Pseudomonas strain LB400 genes encoding polychlorinated biphenyl degradation Mondello,F.J.
  35. Gene v.232 Toluene metabolism by the solvent-tolerant Pseudomonas putida DOT-T1 strain, and its role in solvent impermeabilization Mosqueda,G.;M.I.Ramos-Gonzalez;J.L.Ramos
  36. Mol. Gen. Genet. v.262 A GntR-like negative regulator of the biphenyl degradation genes of the transposon Tn4371 Mouz,S.;C.Merlin;D.Springael;A.Toussaint
  37. J. Microbiol. Biotechnol. v.11 Cloning and phylogenetic analysis fo two different bphC genes and bphD gene from PCB-degrading bacerium, Pseudomonas sp. srtain SY5 Na,K.S.;S.J.Kim;M.Kubo;S.Y.Chung
  38. Proc. Int. conf. Intell. Syst. Mol. Biol. v.6 Prediction of signal peptides and signal anchors by a hidden Markov model Nielsen,H.;A.Krogh
  39. Microbiology v.147 Pseudomonas putida CE2010 can degrade biphenyl by a mosaic pathway encoded by the tod operon and cmtE, which are identical to those of P. putida F1 execpt for a single base difference in the operator-promoter region of the cmt operon Ohta,Y.;M.Maeda;T.Kudo
  40. Gene v.256 Expression of the bph genes involved in biphenyl/PCB degradation in Psudomonas sp. KKS102 induced by the biphenyl degradation intermediate, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid Ohtsubo,Y.;Y.Nagata;K.Kimbara;M.Takagi;A.Ohta
  41. J. Microbiol. Biotechnol. v.9 Induction by carvone of the polychlornated biphenyl (PCB)-degradative pathway in Alcaligenes eutrophus H850 and its molecular monitoring Park,Y.I.;J.S.So;S.C.Koh
  42. Appl. Environ. Microbiol. v.62 Cloning, sequencing, and expression of isopropylbenzene degradation genes from Pseudomonas sp. strain JR1: Indentification of isopropylbenzene dioxygenase that mediates trichloroethene oxidation Pflugmacher,U.;B.Averhoff;G.Gottschalk
  43. J. Baceriol. v.181 Characterization of colicin S4 and its receptor, OmpW, a minor protein of the Escherichia coli outer membrane Pilsl,H.;D.Smajs;V.Braun
  44. Molecular Cloning a Laboratory Manual(3th ed.) Sambrook.J.;D.W.Russell
  45. J. Microbiol. v.39 Physical analysis of nahQ and tnpA genes from Pseudomonas florescens Seol,J.Y.;S.Y.Choi;K.H.Min
  46. J. Bacteriol. v.163 Plasmid-mediated mineralization of 4-chlorobiphenyl Shields,M.S.;S.W.Hooper;G.S.Sayler
  47. Biodegradation v.1 The biodegradation of aromatic hydrocarbons by bacteria Smith,M.R.
  48. Appl. Microbiol. Biotechnol. v.32 Catabolism of alkylbenzenes by Pseudomonas sp. NCIB 10643 Smith,M.R.;C.Ratledge
  49. Appl. Microbiol. Biotechnol. v.30 Catabolism of biphenyl by Pseudomonas sp. NCIB 10643 and Nocardia sp. NCIB 10503 Smith,M.R.;C.Ratledge
  50. Appl. Environ. Microbiol. v.67 Occurrence of Tn4371-related mobile elements and sequences in (chloro)biphenyl-degrading bacteria Springael,D.;A.Ryngaert;C.Merlin;A.Toussaint;M.Mergeay
  51. J. Gen. Microbiol. v.43 The aerobic pseudomonads: a taxomonic study Stanier,R.Y.;N.J.Palleroni;M.Doudoroff
  52. Gene v.196 Green fluorescent protein-based reporter systems for genetic analysis of bacteria including monocopy applications Suarez,A.;A.Guttler;M.Stratz;L.H.Staendner;K.N.Timmis;C.A.Guzman
  53. J. Biol. Chem. v.267 Analysis of bph operon from the polychlorinated biphenyl-degrading strain of Pseudomonas pseudoalcaligenes KF707 Taira,K.;J.Hirose;S.Hayashida;K.Furukawa
  54. Mol. Microbiol. v.6 DNA sequence determination and functional characterization of the OCT- plasmid-encoded alkJKL genes of Pseudomonas oleovorans van Beilen, J. B.;G.Eggink;H.Enequist;R.Bos;B.Witholt
  55. Gene v.33 Improved M13 phage cloning vectors and host strains: Nucleotide sequences of the M13mp18 and pUC19 vectors Yanisch-Perron,C.;J.Vieira;J.Messing
  56. J. Biol. chem. v.264 Toluene degradation by Pseudomonas putida F1. Nucleotide seuqence of the todC1C2BADE genes and their expression in Escherichia coli Zhlstra,G.J.;D.T.Gibson