Skip to main content
SpringerLink
Log in

Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri

  • Original Papers
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

The structural gene, nirK, for the respiratory Cu-containing nitrite reductase from denitrifying Pseudomonas aureofaciens was isolated and sequenced. It encodes a polypeptide of 363 amino acids including a signal peptide of 24 amino acids for protein export. The sequence showed 63.8% positional identity with the amino acid sequence of “Achromobacter cycloclastes” nitrite reductase. Ligands for the blue, type I Cu-binding site and for a putative type-II site were identified. The nirK gene was transferred to the mutant MK202 of P. stutzeri which lacks cytochrome cd 1 nitrite reductase due to a transposon Tn5 insertion in its structural gene, nirS. The heterologous enzyme was active in vitro and in vivo in this background and restored the mutationally interrupted denitrification pathway. Transfer of nirK to Escherichia coli resulted in an active nitrite reductase in vitro. Expression of the nirS gene from P. stutzeri in P. aureofaciens and E. coli led to nonfunctional gene products. Nitrite reductase activity of cell extract from either bacterium could be reconstituted by addition of heme d 1, indicating that both heterologous hosts synthesized a cytochrome cd 1 without the d 1-group.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

Cu-NIR:

Cu-containing nitrite reductase

DDC:

diethyldithiocarbamate

EPR:

electron paramagnetic resonance

IPTG:

isopropyl-β-D-galactoside

SDS:

sodium dodecyl sulfate

LB medium:

Luria-Bertani medium

References

  • Abraham Z, Lowe D, Smith BE (1992) Trimeric nitrite reductase of Achromobacter xylosoxidans has two types of copper centre. J Inorg Chem 47: 45, P01

    Google Scholar 

  • Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41: 459–472

    Google Scholar 

  • Chang CK, Timkovich R, Wu W (1986) Evidence that heme d 1 is a 1,3-porphyrindione. Biochemistry 25: 8447–8453

    Google Scholar 

  • Cole JA (1988) Assimilatory and dissimilatory reduction of nitrate to ammonia. In: Cole JA, Ferguson SJ (eds) The nitrogen and sulphur cycles. Cambridge University Press, Cambridge, pp 281–329

    Google Scholar 

  • Coyle CL, Zumft WG, Kroneck PMH, Körner H, Jakob W (1985) Nitrous oxide reductase from denitrifying Pseudomonas perfectomarina, purification and properties of a novel multicopper enzyme. Eur J Biochem 153: 459–467

    Google Scholar 

  • Coyne MS, Arunakumari A, Averill BA, Tiedje JM (1989) Immunological identification and distribution of dissimilatory heme cd 1 and nonheme copper nitrite reductases in denitrifying bacteria. Appl Environ Microbiol 55: 2924–2931

    Google Scholar 

  • Coyne MS, Arunakumari A, Pankratz HS, Tiedje JM (1990) Localization of the cytochrome cd 1 and copper nitrite reductases in denitrifying bacteria. J Bacteriol 172: 2558–2562

    Google Scholar 

  • Fenderson FF, Kumar S, Adman ET, Liu MY, Payne WJ, LeGall J (1991) Amino acid sequence of nitrite reductase: a copper protein from Achromobacter cycloclastes. Biochemistry 30: 7180–7185

    Google Scholar 

  • Frunzke K, Zumft WG (1984) Rapid, single sample analysis of H2, O2, NO, CO, N2O and CO2 by isothermal gas chromatography: applications to the study of bacterial denitrification. J Chromatogr 299: 477–483

    Google Scholar 

  • Glockner AB, Zumft WG (1992) Primary structure of the copper-containing respiratory nitrite reductase of Pseudomonas aureofaciens and functional expression in Escherichia coli. BioEngineering 8: 76, P424

    Google Scholar 

  • Godden JW, Turley S, Teller DC, Adman ET, Liu MY, Payne WJ, LeGall J (1991) The 2.3 Angstrom X-ray structure of nitrite reductase from Achromobacter cycloclastes. Science 253: 438–442

    Google Scholar 

  • Grisshammer R, Oeckl C, Michel H (1991) Expression in Escherichia coli of c-type cytochrome genes from Rhodopseudomonas viridis. Biochim Biophys Acta 1088: 183–190

    Google Scholar 

  • Grossberger D (1987) Minipreps of DNA from bacteriophage lambda. Nucleic Acids Res 15: 6737

    Google Scholar 

  • Heijne G von (1988) Transcending the impenetrable, how proteins come to terms with membranes. Biochim Biophys Acta 947: 307–333

    Google Scholar 

  • Hill KE, Wharton DC (1978) Reconstitution of the apoenzyme of cytochrome oxidase from Pseudomonas aeruginosa with heme d 1 and other heme groups. J Biol Chem 253: 489–495

    Google Scholar 

  • Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114: 193–197

    Google Scholar 

  • Hulse CL, Tiedje JM, Averill BA (1988) A spectrophotometric assay for dissimilatory nitrite reductases. Anal Biochem 172: 420–426

    Google Scholar 

  • Hulse CL, Averill BA, Tiedje JM (1989) Evidence for a coppernitrosyl intermediate in denitrification by the copper-containing nitrite reductase of Achromobacter cycloclastes. J Am Chem Soc 111: 2322–2323

    Google Scholar 

  • Huynh TV, Young RA, Davis RW (1985) Constructing and screening cDNA libraries in lambda gt10 and gt11. In: Glover DM (ed) DNA cloning, vol. 1. IRL Press Oxford, pp 49–78

    Google Scholar 

  • Iwasaki H, Matsubara T (1972) A nitrite reductase from Achromobacter cycloclastes. J Biochem 71: 645–652

    Google Scholar 

  • Iwasaki H, Shidara S, Suzuki H, Mori T (1963) Studies on denitrification. VII. Further purification and properties of denitrification enzyme. J Biochem 53: 299–303

    Google Scholar 

  • Jackson RH, Cornish-Bowden A, Cole JA (1981) Prosthetic groups of the NADH-dependent nirite reductase from Escherichia coli K12. Biochem J 193: 861–867

    Google Scholar 

  • Jüngst A, Braun C, Zumft WG (1991a) Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification. Mol Gen Gent 225: 241–248

    Google Scholar 

  • Jüngst A, Zumft WG (1992) Heterologous expression of respiratory nitrite reductase (cytochrome cd 1) of Pseudomonas stutzeri in Escherichia coli and Pseudomonas aureofaciens. BioEngineering 8: 76, P423

    Google Scholar 

  • Jüngst A, Wakabayashi S, Matsubara H, Zumft WG (1991b) The nirSTBM region coding for cytochrome cd 1-dependent nitrite respiration of Pseudomonas stutzeri consists of a cluster of mono-, di-, and tetraheme proteins. FEBS Lett 279: 205–209

    Google Scholar 

  • Kajie S, Anraku Y (1986) Purification of a hexaheme cytochrome c 552 from Escherichia coli K12 and its properties as a nitrite reductase. Eur J Biochem 154: 457–463

    Google Scholar 

  • Kakutani T, Watanabe H, Arima K, Beppu T (1981) A blue protein as an inactivating factor for nitrite reductase from Alcaligenes faecalis strain S-6. J Biochem 89: 463–472

    Google Scholar 

  • Körner H, Mayer F (1992) Periplasmic location of nitrous oxide reductase and its apoform in denitrifying Pseudomonas stutzeri. Arch Microbiol 157: 218–222

    Google Scholar 

  • Körner H, Frunzke K, Döhler K, Zumft WG (1987) Immunochemical patterns of distribution of nitrous oxide reductase and nitrite reductase (cytochrome cd 1) among denitrifying pseudomonads. Arch Microbiol 148: 20–24

    Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London) 227: 680–685

    Google Scholar 

  • Laurell CB, McKay EJ (1981) Electroimmunoassay. Meth Enzymol 73: 339–369

    Google Scholar 

  • Libby E, Averill BA (1992) Evidence that the type 2 copper centers are the site of nitrite reduction by Achromobacter cycloclastes nitrite reductase. Biochem Biophys Res Commun 187: 1529–1535

    Google Scholar 

  • Liu MC, Payne WJ, Peck HD, Jr, LeGall J (1983) Comparison of cytochromes from anaerobically and aerobically grown cells of Pseudomonas perfectomarinus. J Bacteriol 154: 278–286

    Google Scholar 

  • Liu MY, Liu MC, Payne WJ, LeGall J (1986) Properties and electron transfer specifity of copper proteins from the denitrifier Achromobacter cycloclastes. J Bacteriol 166: 604–608

    Google Scholar 

  • Masuko M, Iwasaki H, Sakurai T, Suzuki S, Nakahara A (1984) Characterization of the nitrite reductase from a denitrifier, Alcaligenes Sp. NCIB 11015. A novel copper protein. J Biochem 96: 447–454

    Google Scholar 

  • Matsubara T, Zumft WG (1982) Identification of a copper protein as part of the nitrous oxide-reducing system in nitrite-respiring (denitrifying) pseudomonads. Arch Microbiol 132: 322–328

    Google Scholar 

  • Messing J, Gronenborn B, Müller-Hill B, Hofschneider PH (1977) Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci USA 74: 3642–3646

    Google Scholar 

  • Page MD, Ferguson SJ (1989) A bacterial c-type cytochrome can be translocated to the periplasm as an apoform: the biosynthesis of cytochrome cd 1 (nitrite reductase) from Paracoccus denitrificans. Mol Microbiol 3: 653–661

    Google Scholar 

  • Priefer UB, Simon R, Pühler A (1985) Extension of the host range of Escherichia coli vectors by incorporation of RSF1010 replication and mobilization functions. J Bacteriol 163: 324–330

    Google Scholar 

  • Ryden L, Lundgren JO (1976) Homology relationships among the small blue proteins. Nature 261: 344–346

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY

    Google Scholar 

  • Shapleigh JP, Payne WJ (1985) Differentation of c, d 1 cytochrome and copper nitrite reductase production in denitrifiers. FEMS Microbiol Lett 26: 275–279

    Google Scholar 

  • Silvestrini MC, Cutruzzola F, D'Alessandro R, Brunori M, Fochesato N, Zennaro E (1992) Expression of Pseudomonas aeruginosa nitrite reductase in Pseudomonas putida and characterization of the recombinant protein. Biochem J 285: 661–666

    Google Scholar 

  • Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in Gram-negative bacteria. Bio/Technology 1: 784–791

    Google Scholar 

  • Studier FW, Moffatt BA (1986) Use of bacteriophage T7 RNA polymerase to direct selective high-level expression of cloned genes. J Mol Biol 189: 113–130

    Google Scholar 

  • Suzuki S, Yoshimura T, Kohzuma T, Shidara S, Masuko M, Sakurai T, Iwasaki H (1989) Spectroscopic evidence for a coppernitrosyl intermediate in nitrite reduction by blue copper-containing nitrite reductase. Biochem Biophys Res Commun 104: 1366–1372

    Google Scholar 

  • Thomas PE, Ryan D, Levin W (1976) An improved staining procedure for the detection of peroxidase activity of cytochrome P-450 on sodium dodecyl sulfate polyacrylamide gels. Anal Biochem 75: 168–176

    Google Scholar 

  • Timkovich R, Cork MS, Gennis RB, Johnson PY (1985) Proposed structure of heme d, a prosthetic group of bacterial terminal oxidases. J Am Chem Soc 107: 6096–6075

    Google Scholar 

  • Wachenfeldt C von, Hederstedt L (1990) Bacillus subtilis holocytochrome c-550 can be synthesized in aerobic Escherichia coli. FEBS Lett 270: 147–151

    Google Scholar 

  • Yamanaka T, Kijimoto S, Okunuki K (1963) Biological significance of Pseudomonas cytochrome oxidase in Pseudomonas aeruginosa. J Biochem 53: 416–421

    Google Scholar 

  • Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33: 103–119

    Google Scholar 

  • Young RA, Davis RW (1983a) Yeast RNA polymerase II genes: isolation with antibody probes. Science 222: 778–782

    Google Scholar 

  • Young RA, Davis RW (1983b) Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 89: 1194–1198

    Google Scholar 

  • Young RA, Bloom BR, Crossinsky CM, Ivanyi J, Thomas D, Davis RW (1985) Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc Natl Acad Sci USA 82: 2583–2587

    Google Scholar 

  • Zumft WG (1992) The denitrifying prokaryotes. In: Balows A, Trüper HG, Dworkin M, Harder W, Schleifer KH (eds) The prokaryotes. A handbook on the biology of bacteria: ecophysiology, isolation, identification, applications, 2nd ed, vol. 1. Springer Berlin, Heidelberg, New York, pp 554–582

    Google Scholar 

  • Zumft WG, Döhler K, Körner H (1985) Isolation and characterization of transposon Tn5-induced mutants of Pseudomonas perfectomarina defective in nitrous oxide respiration. J Bacteriol 163: 918–924

    Google Scholar 

  • Zumft WG, Gotzman DJ, Kroneck PMH (1987) Type 1, blue copper proteins constitute a respiratory nitrite-reducing system in Pseudomonas aureofaciens. Eur J Biochem 168: 301–307

    Google Scholar 

  • Zumft WG, Döhler K, Körner H, Löchelt S, Viebrock A, and Frunzke K (1988) Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri. Arch Microbiol 149: 492–498

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Lehrstuhl für Mikrobiologie, Universität Karlsruhe, Kaiserstrasse 12, D-76 128, Karlsruhe, Germany

    Andrea B. Glockner, Angelika Jüngst & Walter G. Zumft

Authors
  1. Andrea B. Glockner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Angelika Jüngst
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Walter G. Zumft
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Glockner, A.B., Jüngst, A. & Zumft, W.G. Copper-containing nitrite reductase from Pseudomonas aureofaciens is functional in a mutationally cytochrome cd 1-free background (NirS−) of Pseudomonas stutzeri . Arch. Microbiol. 160, 18–26 (1993). https://doi.org/10.1007/BF00258141

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00258141

Key words

Search

Navigation