Abstract
Pseudomonas putida GJ31 harbors a degradative pathway for chlorobenzene via meta-cleavage of 3-chlorocatechol. Pseudomonads using this route for chlorobenzene degradation, which was previously thought to be generally unproductive, were isolated from various contaminated environments of distant locations. The new isolates, Pseudomonas fluorescens SK1 (DSM16274), Pseudomonas veronii 16-6A (DSM16273), Pseudomonas sp. strain MG61 (DSM16272), harbor a chlorocatechol 2,3-dioxygenase (CbzE). The cbzE-like genes were cloned, sequenced, and expressed from the isolates and a mixed culture. The chlorocatechol 2,3-dioxygenases shared 97% identical amino acids with CbzE from strain GJ31, forming a distinct family of catechol 2,3-dioxygenases. The chlorocatechol 2,3-dioxygenase, purified from chlorobenzene-grown cells of strain SK1, showed an identical N-terminal sequence with the amino acid sequence deduced from cloned cbzE. In all investigated chlorobenzene-degrading strains, cbzT-like genes encoding ferredoxins are located upstream of cbzE. The sequence data indicate that the ferredoxins are identical (one amino acid difference in CbzT of strain 16-6A compared to the others). In addition, the structure of the operon downstream of cbzE is identical in strains GJ31, 16-6A, and SK1 with genes cbzX (unknown function) and the known part of cbzG (2-hydroxymuconic semialdehyde dehydrogenase) and share 100% nucleotide sequence identity with the entire downstream region. The current study suggests that meta-cleavage of 3-chlorocatechol is not an atypical pathway for the degradation of chlorobenzene.
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Alfreider A, Vogt C, Babel W (2003) Expression of chlorocatechol 1,2-dioxygenase and chlorocatechol 2,3-dioxygenase genes in chlorobenzene-contaminated subsurface samples. Appl Environ Microbiol 69:1372–1376
Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402
Bartels I, Knackmuss H-J, Reineke W (1984) Suicide inactivation of catechol 2,3-dioxygenase from Pseudomonas putida mt-2 by 3-halocatechols. Appl Environ Microbiol 47:500–550
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248-254
Dorn E, Hellwig M, Reineke W, Knackmuss H-J (1974) Isolation and characterization of a 3-chlorobenzoate degrading pseudomonad. Arch Microbiol 99:61–70
Edwards U, Rogall T, Blöcker H, Emde M, Böttger EC (1989) Isolation and direct complete nucleotide determination of entire genes. Characterization of a gene coding for 16S ribosomal RNA. Nucleic Acids Res 17:7843–7853
Eltis LD, Bolin JT (1996) Evolutionary relationships among extradiol dioxygenases. J Bacteriol 178:5930–5937
Harayama S, Rekik M (1990) The meta cleavage operon of TOL degradative plasmid pWW0 comprises 13 genes. Mol Gen Genet 221:113–120
Harayama S, Polissi A, Rekik M (1991) Divergent evolution of chloroplast-type ferredoxins. FEBS Lett 285:85–88
Higgins DG, Bleasby AJ, Fuchs R (1992) CLUSTAL V: improved software for multiple sequence alignment. Comput Appl Biosci 8:189–191
Kaschabek SR, Kasberg T, Müller D, Mars AE, Janssen DB, Reineke W (1998) Degradation of chloroaromatics: purification and characterization of a novel type of chlorocatechol 2,3-dioxygenase of Pseudomonas putida GJ31. J Bacteriol 180:296–302
King EO, Ward MK, Raney DE (1954) Two simple media for the demonstration of pyocyanin and fluorescin. J Lab Clin Med 44:301–307
Klecka GM, Gibson DT (1981) Inhibition of catechol 2,3-dioxygenase from Pseudomonas putida by 3-chlorocatechol. Appl Environ Microbiol 41:1159–1165
Knackmuss H-J (1981) Degradation of halogenated and sulfonated hydrocarbons. In: Leisinger T, Hütter R, Cook AM, Nüesch J (eds) Microbial degradation of xenobiotics and recalcitrant compounds. Acadamic, London, pp 190–212
Lane DJ, Pace B, Olsen GJ, Stahl DA, Sogin ML, Pace NR (1985) Rapid determination of 16S ribosomal RNA sequences for phylogenetic analyses. Proc Natl Acad Sci USA 82:6955–6959
Mars AE, Kasberg T, Kaschabek SR, van Agteren MH, Janssen DB, Reineke W (1997) Microbial degradation of chloroaromatics: use of the meta-cleavage pathway for mineralization of chlorobenzene. J Bacteriol 179:4530–4537
Mars AE, Kingma J, Kaschabek SR, Reineke W, Janssen DB (1999) Conversion of 3-chlorocatechol by various catechol 2,3-dioxygenases and sequence analysis of the chlorocatechol dioxygenase region of Pseudomonas putida GJ31. J Bacteriol 181:1309–1318
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor
Moos M Jr (1993) Isolation of proteins for microsequence analysis. In: Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman G, Smith JA, Struhl K (eds) Current protocols in molecular biology, supplement 21, Wiley, New York, pp 10.19.1–10.19.12
Nakai C, Kagamiyama H, Nozaki M, Nakazawa T, Inouye S, Ebina Y, Nakazawa A (1983) Complete nucleotide sequence of the metapyrocatechase gene on the TOL plasmid of Pseudomonas putida mt-2. J Biol Chem 258:2923–2928
Oldenhuis R, Kuijk L, Lammers A, Janssen DB, Witholt B (1989) Degradation of chlorinated and non-chlorinated aromatic solvents in soil suspensions by pure bacterial cultures. Appl Microbiol Biotechnol 30:211–217
Page RDM (1996) TREEVIEW: an application to display phylogenetic trees on personal computers. Comput Appl Biosci 12:357–358
Parales RE, Ontl TA, Gibson DT (1997) Cloning and sequence analysis of a catechol 2,3-dioxygenase gene from the nitrobenzene-degrading strain Comamonas sp. JS765. J Ind Microbiol Biotechnol 19:385–391
Pearson WR, Lipman DJ (1988) Improved tools for biological sequence comparison. Proc Natl Acad Sci USA 85:2444–2448
Reineke W (1998) Development of hybrid strains for the mineralization of chloroaromatics by patchwork assembly. Annu Rev Microbiol 52:287–331
Riegert U, Heiss G, Kuhm AE, Müller C, Contzen M, Knackmuss H-J, Stolz A (1999) Catalytic properties of the 3-chlorocatechol-oxidizing 2,3-dihydroxybiphenyl 1,2-dioxygenase from Sphingomonas sp. strain BN6. J Bacteriol 181:4812–4817
Riegert U, Bürger S, Stolz A (2001) Altering catalytic properties of 3-chlorocatechol-oxidizing extradiol dioxygenase from Sphingomonas xenophaga BN6 by random mutagenesis. J Bacteriol 183:2322–2330
Saint CP, McClure NC, Venables WA (1990) Physical map of the aromatic amine and m-toluate catabolic plasmid pTDN1 in Pseudomonas putida: localization of a unique meta-cleavage pathway. J Gen Microbiol 136:615–625
Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring Harbor, New York
Schlömann M (1994) Evolution of chlorocatechol catabolic pathways. Conclusions to be drawn from comparisons of lactone hydrolases. Biodegradation 5:301–321
Schreiber A, Hellwig M, Dorn E, Reineke W, Knackmuss H-J (1980) Critical reactions in fluorobenzoic acid degradation by Pseudomonas sp. B13. Appl Environ Microbiol 39:58–67
Shingler V, Powlowski J, Marklund U (1992) Nucleotide sequence and functional analysis of the complete phenol/3,4-dimethylphenol catabolic pathway of Pseudomonas sp. strain CF600. J Bacteriol 174:711–724
Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680
Tropel D, Meyer C, Armengaud J, Jouanneau Y (2002) Ferredoxin-mediated reactivation of the chlorocatechol 2,3-dioxygenase from Pseudomonas putida GJ31. Arch Microbiol 177:345–351
Vaillancourt FH, Labbé G, Drouin NM, Fortin PD, Eltis LD (2002) The mechanism-based inactivation of 2,3-dihydroxybiphenyl 1,2-dioxygenase by catecholic substrates. J Biol Chem 277:2019–2027
Volff J-N, Eichenseer C, Viell P, Piendl W, Altenbuchner J (1996) Nucleotide sequence and role in DNA amplification of the direct repeats composing the amplificable element AUD1 of Streptomyces lividans 66. Mol Microbiol 21:1037–1047
Acknowledgments
This work was financed by a grant from the Deutsche Forschungsgemeinschaft (Re659/7-1) and by the European Union, contract EVK1-CT1999-00023 “MAROC”. The MAROC samples were supplied by Bioclear, Groningen, The Netherlands. We thank Y. Jouanneau, Grenoble, France, for critical reading of the manuscript, U. Pagga, BASF, Ludwigshafen, Germany, for a waste water sample, N.C. McClure, Flinders University of South Australia, Adelaide, Australia, for sending pTDN1-1018, and V. Nödinger, University Stuttgart, Germany, for the amino acid sequencing. We are indebted to CEA for providing the environment in which O.K. could do his work in Grenoble.
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This publication is dedicated to the memory of Olga V. Maltseva, who contributed greatly to our current knowledge of biochemistry of degradative pathways for chloroaromatic compounds.
This publication is dedicated to Prof. Dr. Hans G. Schlegel in honor of his 80th birthday.
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Göbel, M., Kranz, O.H., Kaschabek, S.R. et al. Microorganisms degrading chlorobenzene via a meta-cleavage pathway harbor highly similar chlorocatechol 2,3-dioxygenase-encoding gene clusters. Arch Microbiol 182, 147–156 (2004). https://doi.org/10.1007/s00203-004-0681-5
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DOI: https://doi.org/10.1007/s00203-004-0681-5