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  2. Abstract

The structural gene for the respiratory nitrous-oxide reductase from Paracoccus denitrificans has been cloned using a probe derived from the structural gene, nosZ, for this enzyme from Pseudomonas stutzeri. The cloned gene could be expressed surprisingly well (presumably yielding an apo-protein) using an expression vector in Escherichia coli.

Sequencing the nosZ gene from P. denitrificans has shown that the periplasmic nitrous-oxide reductase of this organism is highly similar in sequence to previously derived primary sequences for the enzyme from three other organisms. As with the other reductases, an unusually long signal sequence is deduced and a common motif of GXXRRXXLG near the beginning of this sequence is present. The results of N-terminal sequencing of the mature nitrous-oxide reductase from the closely related organism Thiosphaera pantotropha indicate that processing of the P. denitrificans precursor occurs between amino acids at positions 57 and 58. The predicted signal peptide is therefore of the same length and of similar overall structure to that previously described for the P. denitrificans methylamine dehydrogenase small subunit (MauA). The P. denitrificans sequence for the mature nitrous-oxide reductase reduces from 14 to 11 and 6 to 4, respectively, the number of conserved histidine and methionine residues compared to previous sequences. Three cysteine and four tryptophan residues, previously identified as conserved amongst nitrous-oxide reductases, are found in the Paracoccus enzyme. A comparison of the sequence of the C-terminal region of the nitrous-oxide-reductase sequence with that for the CuA region of subunit II of the cytochrome aa3 from P. denitrificans reveals considerable sequence similarities.

Upstream of the structural gene for nosZ are sequences TTGAAGCTTAACCAG (centred at position –21 with respect to the start codon) and CCCGGTGGTCATCAAG (centred at position –126). Although both could be FNR (ANR) boxes, the latter is far more probable to have this role because only it is likely to be upstream of a promoter site. This is the first indication at the DNA sequence level for the existence of this regulatory system in P. denitrificans.

Analysis of the flanking DNA sequences revealed reading frames upstream and downstream of the nosZ gene showing similarity to the nosR and nosD genes, respectively, of Pseudomonas species.

An S30 in vitro transcription/translation system was developed for P. denitrificans which permitted the expression of the cloned gene for nitrous-oxide reductase and which will be of general value in other studies of this organism.

FNR box

binding site for a trans-acting regulatory protein (Fnr) that activates gene expression under anaerobic conditions

ANR box

the equivalent to FNR box in P. aeruginosa


Nitrous-oxide reductase (EC


  1. Top of page
  2. Abstract
  • Alefounder, P. R., Greenfield, A. J., McCarthy, J. E. G. & Ferguson, S. J. (1983) Selection and organization of denitrifying electrontransfer pathways in Paracoccus denitrificans, Biochim. Biophys. Acta 723, 2039.
  • Antholine, W. E., Kastrau, D. H. W., Steffens, G. C. M., Buse, G., Zumft, W. G. & Kroneck, P. M. H. (1992) A comparative EPR investigation of the multicopper proteins nitrous-oxide reductase and cytochrome c oxidase, Eur. J. Biochem. 209, 875881.
  • Berks, B. C., Baratta, D., Richardson, D. J. & Ferguson, S. J. (1993) Purification and characterization of a nitrous oxide reductase from Thiosphaera pantotropha. Implications for the mechanism of aerobic nitrous oxide reduction, Eur. J. Biochem. 212, 467476.
  • Boogerd, F. C., van Verseveld, H. W. & Stouthamer, A. H. (1981) Respiration-driven proton translocation with nitrite and nitrous oxide in Paracoccus denitrificans, Biochim. Biophys. Acta 638, 181191.
  • Burnell, J. N., John, P. & Whatley, F. R. (1978) The reversibility of active sulphate transport into membrane vesicles of Paracoccus denitrificans, Biochem. J. 150, 527536.
  • Chistoserdov, A. Y. & Lidstrom, M. E. (1991) The small-subunit polypeptide of methylamine dehydrogenase from Methylobacterium extorquens AMI has an unusual leader sequence, J. Bacteriol. 173, 59095913.
  • Chistoserdov, A. Y., Boyd, J., Mathews, F. S. & Lidstrom, M. (1992) The genetic organization of the Mau gene cluster of the facultative autotroph Paracoccus denitrificans, Biochem. Biophys. Res. Commun. 184, 11811189.
  • Coyle, C. L., Zumft, W. G., Kroneck, P. M. H., Korner, H. & Jakob, W. (1985) Nitrous-oxide reductase from denitrifying Pseudomonas perfectomarina, purification, and properties of a novel multicopper enzyme, Eur. J. Biochem. 153, 459467.
  • Cuypers, H. & Zumft, W. G. (1992) Regulatory components of the denitrification gene cluster of Pseudomonas stutzeri in Pseudomonas: molecular biology and biotechnology (Galli, E., Silver, S. & Witholt, B., eds) pp. 188197, American Society for Microbiology, Washington , DC .
  • Cuypers, H., Viebrock-Sambale, A. & Zumft, W. G. (1992) NosR, a membrane-bound regulatory component necessary for expression of nitrous oxide reductase in denitrifying Pseudomonas stutzeri, J. Bacteriol. 174, 53325339.
  • De Vries, G. E., Harms, N., Hoogendijk, J. & Stouthamer, A. H. (1989) Isolation and characterization of Paracoccus denitrificans mutants with increased conjugation frequencies and pleiotropic loss of a (nGATCn) DNA-modifying property, Arch. Microbiol. 152, 5257.
  • Ehrich, E., Craig, A., Poustka, A., Frischauf, A. M. & Lehrach, H. (1987) A family of cosmid vectors with the multicopy R6K replication origin, Gene (Amst.) 57, 229237.
  • Farrar, J., Thomson, A. J., Cheesman, M. R., Dooley, D. M. & Zumft, W. G. (1991) A model of the copper centres of nitrous-oxide reductase (Pseudomonas stutzeri). Evidence from optical, EPR and MCD spectroscopy, FEBS Lett. 294, 1115.
  • Ferguson, S. J. (1987) Denitrification: a question of the control and organisation of electron and ion transport, Trends Biochem. Sci. 12, 354357.
  • Gallimand, M., Gamper, M., Zimmermann, A. & Haas, D. (1991) Positive FNR-like control of anaerobic arginine degradation and nitrate respiration in Pseudomonas aeruginosa, J. Bacteriol. 173, 15981606.
  • Gottesman, M. E., Adhya, S. & Das, A. (1980) Transcription antitermination by bacteriophage λN gene product, J. Mol. Biol. 140, 5775.
  • Guest, J. R. (1992) Oxygen-regulated gene expression in E. coli, J. Gen. Microbiol. 138, 22532263.
  • Harms, N., de Vries, G. E., Maurer, K., Hoogendijk, J. & Stouthamer, A. H. (1989) Isolation and nucleotide sequence of the methanol dehydrogenase structural gene from Paracoccus denitrificans, J. Bacteriol. 169, 39693975.
  • Henikoff, S. (1984) Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA-sequencing. Gene (Amst.) 28, 351359.
  • Holm, L., Saraste, M. & Wikstrom, M. (1987) Structural models for the redox centres in cytochrome oxidase, EMBO J. 6, 28192823.
  • Klionsky, R. J., Brusilow, W. S. A. & Simoni, R. D. (1984) In vivo evidence for the role of the ɛ subunit as an inhibitor of the proton-translocating ATPase of E. coli, J. Bacteriol. 160, 10551060.
  • Korner, H. & Zumft, W. G. (1989) Expression of denitrification enzymes in response to dissolved oxygen level and respiratory substrate in continuous culture of Pseudomonas stutzeri, Appl. Environ. Microbiol. 55, 16701676.
  • Kucera, I., Boublikova, P. & Dadak, V. (1984) Function of terminal acceptors in the biosynthesis of denitrification pathway components in Paracoccus denitrificans, Folia Microbiol. 29, 108114.
  • Ludwig, W., Mittenhuber, G. & Friedrich, C. G. (1993) Transfer of Thiosphaera pantotropha to Paracoccus denitrificans, Int. J. Syst. Bacteriol. 43, 363367.
  • McEwan, A. G., Greenfield, A. J., Wetzstein, H. G., Jackson, J. B. & Ferguson, S. J. (1985) Nitrous-oxide reduction by members of the family Rhodospirillaceae and the nitrous-oxide reductase Rhodopseudomonas capsulata, J. Bacteriol. 164, 823830.
  • Nicholls, D. G. & Ferguson, S. J. (1992) Bioenergetics 2, Academic Press, London and New York .
  • Page, M. D., Carr, G., Bell, L. C. & Ferguson, S. J. (1989) The structure, control and assembly of a bacterial electron transport system as exemplified by Paracoccus denitrificans, Biochem. Soc. Trans. 17, 991993.
  • Pratt, C. (1980) Kinetics and regulation of cell-free alkaline phosphatase synthesis. J. Bacteriol. 143, 12651274.
  • Raitio, M., Jalli, T. & Saraste, M. (1987) Isolation and analysis of the genes for cytochrome oxidase in Paracoccus denitrificans, EMBO J. 6, 28252833.
  • Sambrook, J., Fritsch, E. F. & Maniatis, T. (1992) Molecular cloning: a laboratory manual, 2nd edn, Cold Spring Harbor Laboratory, Cold Spring Harbor , NY .
  • Samyn, B., Berks, B. C., Page, M. L. D., Ferguson, S. J. & van Beeumen, J. J. (1993) Characterization and complete amino acid sequence of cytochrome c-550 from Thiosphaera pantotropha, Eur. J. Biochem., in the press.
  • Sanger, F., Nicklen, S. & Coulson, A. R. (1977) DNA sequencing with chain-terminating inhibitors, Proc. Natl Acad. Sci USA 74, 54635467.
  • Sawers, R. G. (1991) Identification and molecular characterization of a transcriptional regulator from Pseudomonas aeruginosa PA01 exhibiting structural and functional similarity to the FNR protein of Escherichia coli, Mol. Microbiol. 5, 14691481.
  • Schatz, G. (1993) The protein import machinery of mitochondria, Protein Science 2, 141146.
  • Schauder, B., Blöcker, H., Frank, R. & McCarthy, J. E. G. (1987) Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region, Gene (Amst.) 52, 279283.
  • Snyder, S. W. & Hollocher, T. C. (1987) Purification and some characteristics of nitrous-oxide reductase from Paracoccus denitrificans, J. Biol. Chem. 262, 65156525.
  • Spiro, S. (1992) An FNR-dependent promoter from Escherichia coli is active and anaerobically inducible in Paracoccus denitrificans, FEMS Microbiol. Lett. 98, 145148.
  • Spiro, S. & Guest, J. (1990) FNR and its role in oxygen-regulated gene expression in Escherichia coli. FEMS Microbiol. Rev. 75, 399428.
  • Steinrücke, P. & Ludwig, B. (1993) Genetics of Paracoccus denitrificans, FEMS Microbiol. Rev. 104, 83118.
  • Stouthamer, A. H. (1992) Metabolic pathways in Paracoccus denitrificans and closely related bacteria in relation to the phylogeny of prokaryotes, Antonie Leeuwenhoek 61, 133.
  • Van Spanning, R. J. M., Wansell, C., Harms, N., Oltmann, L. F. & Stouthamer, A. H. (1990a) Mutagenesis of the gene encoding cytochrome c550 of Paracoccus denitrificans and analysis of resultant physiological effects, J. Bacteriol. 172, 986996.
  • Van Spanning, R. J. M., Wansell, C. W., Reijnders, W. N. M., Oltmann, L. F. & Stouthamer, A. H. (1990b) Mutagenesis of the gene encoding amicyanin of Paracoccus denitrificans and the resultant effect on methylamine oxidation, FEBS Lett. 275, 217220.
  • Viebrock, A. & Zumft, W. G. (1988) Molecular cloning, heterologous expression, and primary structure of the structural gene for the copper enzyme nitrous-oxide reductase from denitrifying Pseudomonas stutzeri, J. Bacteriol. 170, 46584668.
  • von Heijne, G. (1987) Sequence analysis molecular biology: treasure trove or trivial pursuit, Academic Press, London and New York .
  • Zubay, G. (1973) In vitro synthesis of protein in microbial systems, Annu. Rev. Genet. 7, 267287.
  • Zumft, W. G., Viebrock-Sambale, A. & Braun, C. (1990) Nitrous-oxide reductase from denitrifying Pseudomonas stutzeri. Eur. J. Biochem. 192, 591599.
  • Zumft, W. G. & Matsubara, T. (1982) A novel kind of multi-copper protein as terminal oxidoreductase of nitrous-oxide respiration in Pseudomonas stutzeri, FEBS Lett. 148, 107112.
  • Zumft, W. G. (1992) The denitrifying prokaryotes in The prokaryotes. A handbook on the biology of bacteria, ecophysiology, isolation, identification, applications (Balows, A., Truper, H. G., Dworkin, M., Harder, W. & Schleifer, K., eds) pp. 554582, Springer-Verlag, New York .
  • Zumft, W. G., Dreusch, A., Lochelt, S., Cuypers, H., Friedrich, B. & Schneider, B. (1992) Derived amino acid sequences of the nos Z gene (respiratory N2O reductase) from Alcaligenes eutrophus, Pseudomonas aeruginosa and Pseudomonas stutzeri reveal potential copper-binding residues implications for the CuA site of N2O reductase and cytochrome-c oxidase, Eur. J. Biochem. 208, 3140.