Advertisement

Molecular and General Genetics MGG

, Volume 225, Issue 2, pp 241–248 | Cite as

Close linkage in Pseudomonas stutzeri of the structural genes for respiratory nitrite reductase and nitrous oxide reductase, and other essential genes for denitrification

  • Angelika Jüngst
  • Cornelia Braun
  • Walter G. Zumft
Article

Summary

The structural gene, nirS, for the respiratory nitrite reductase (cytochrome cd1) from Pseudomonas stutzeri was identified by (i) sequencing of the N-terminus of the purified protein and partial sequencing of the cloned gene, (ii) immunoscreening of clones from a lambda gt11 expression library, (iii) mapping of the transposon Tn5 insertion site in the nirS mutant strain MK202, and (iv) complementation of strain MK202 with a plasmid carrying the insert from an immunopositive lambda clone. A mutation causing overproduction of cytochrome c552 mapped on the same 8.6 kb EcoRI fragment within 1.7 kb of the mutation affecting nirS. Two mutations affecting nirD, which cause the synthesis of an inactive cytochrome cd1 lacking heme d1, mapped 1.1 kb apart within a 10.5 kb EcoRI fragment contiguous with the fragment carrying nirS. Nir mutants of another type that had low level synthesis of cytochrome cd1, had Tn5 insertions within an 11 kb EcoRI fragment unlinked to the nirS+ and nirD+ fragments. Cosmid mapping provided evidence that nirS and nirD, and the previously identified gene cluster for nitrous oxide respiration are closely linked. The nirS gene and the structural gene for nitrous oxide reductase, nosZ, are transcribed in the same direction and are separated by approximately 14 kb. Several genes for copper processing are located within the intervening region.

Key words

Bacterial denitrification Cytochrome cd1 Nitrous oxide respiration Transposon Tn5 Gene cluster 

Preview

Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

References

  1. Arai H, Sanbongi Y, Igarashi Y, Kodama T (1990) Cloning and sequencing of the gene encoding cytochrome c 551 from Pseudomonas aeruginosa. FEBS Lett 261:196–198Google Scholar
  2. Bolivar F (1978) Construction and characterization of new cloning vehicles. III. Derivatives of plasmid pBR322 carrying unique EcoRI sites for selection of EcoRI generated recombinant DNA molecules. Gene 4:121–136Google Scholar
  3. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472Google Scholar
  4. Chang CK, Timkovich R, Wu W (1986) Evidence that heme d1 is a 1,3-porphyrindione. Biochemistry 25:8447–8453Google Scholar
  5. Chu G, Vollrath D, Davis RW (1986) Separation of large DNA molecules by contour-clamped homogeneous electric fields. Science 234:1582–1585Google Scholar
  6. 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 153:459–467Google Scholar
  7. 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 55:2924–2931Google Scholar
  8. De Blas AL, Cherwinski HM (1983) Detection of antigens on nitrocellulose paper. Immunoblots with monoclonal antibodies. Anal Biochem 133:214–219Google Scholar
  9. Döhler K, Huss VAR, Zumft WG (1987) Transfer of Pseudomonas perfectomarina Baumann, Bowditch, Baumann, and Beaman 1983 to Pseudomonas stutzeri (Lehman and Neumann 1896) Sijderius 1946. Int J Syst Bacteriol 37:1–3Google Scholar
  10. Frantz B, Chakrabarty AM (1986) Degradative plasmids in Pseudomonas. In: Sokatch JR (ed) The bacteria. A treatise on structure and function, vol 10. The biology of Pseudomonas. Academic Press, Orlando, pp 295–323Google Scholar
  11. Friedrich B, Böcker C, Eberz G, Eitinger T, Horstmann K, Kortlüke C, Römermann D, Schwartz E, Tran-Betcke A, Warnecke U, Warrelmann J (1990) Genes for hydrogen oxidation and denitrification form two clusters on megaplasmid pHG1 of Alcaligenes eutrophus. In: Silver S, Chakrabarty AM, Iglewski B, Kaplan S (eds) Pseudomonas. Biotransformations; pathogenesis, and evolving biotechnology. American Society for Microbiology, Washington, DC, pp 408–419Google Scholar
  12. Grossberger D (1987) Minipreps of DNA from bacteriophage lambda. Nucleic Acids Res 15:6737Google Scholar
  13. Henry Y, Bessières P (1984) Denitrification and nitrite reduction: Pseudomonas aeruginosa nitrite-reductase. Biochimie 66:259–289Google Scholar
  14. Hohn B (1979) In vitro packaging of lambda and cosmid DNA. Methods Enzymol 68:299–309Google Scholar
  15. Holloway BW, Morgan AF (1986). Genome organization in Pseudomonas. Annu Rev Microbiol 40:79–105Google Scholar
  16. Holmes DS, Quigley M (1981) A rapid boiling method for the preparation of bacterial plasmids. Anal Biochem 114:193–197Google Scholar
  17. Huynh TV, Young RA, Davis RW (1985) Constructing and screening cDNA libraries in lambda gt10 and lambda gt11. In: Glover DM (ed) DNA cloning, vol 1. IRL Press, Oxford, pp 49–78Google Scholar
  18. Jayaraman PS, Peakman TC, Busby SWJ, Quincey RV, Cole JA (1987) Location and sequence of the promoter of the gene for the NADH-dependent nitrite reductase of Escherichia coli and its regulation by oxygen, the Fnr protein and nitrite. J Mol Biol 196:781–788Google Scholar
  19. Jeter RM, Ingraham JL (1981) The denitrifying prokaryotes. In: Starr MP, Stolp H, Trüper HG, Balows A, Schlegel HG (eds) The prokaryotes: a handbook on habitats, isolation and identification of bacteria, vol 1. Springer-Verlag, Berlin, pp 913–925Google Scholar
  20. Eingst A, Braun C, Zumft WG (1990) Structural genes for nitrite reductase (cytochrome cd 1) and nitrous oxide reductase are part of a gene cluster. Forum Mikrobiol 13:55 (Abstract)Google Scholar
  21. Kroneck PMH, Antholine WA, Riester J, Zumft WG (1988) The cupric site in nitrous oxide reductase contains a mixed-valence [Cu(II), Cu(I)] binuclear center: a multifrequency electron paramagnetic resonance investigation. FEBS Lett 242:70–74Google Scholar
  22. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685Google Scholar
  23. Leary JJ, Brigati DJ, Ward DC (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitro-cellulose: bioblots. Proc Natl Acad Sci USA 80:4045–4049Google Scholar
  24. Leidigh BJ, Wheelis ML (1973) The clustering on the Pseudomonas putida chromosome of genes specifying dissimilatory functions. J Mol Evol 2:235–242Google Scholar
  25. Lindenmaier W (1985) Vektor-Wirt Systeme zur DNA-Klonierung in E. coli. In: Blin N, Trendelenburg MF, Schmidt ER (eds) Molekular-und Zellbiologie. Springer-Verlag, Berlin, pp 65–85Google Scholar
  26. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  27. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New YorkGoogle Scholar
  28. Meade HM, Long SR, Ruvkun GB, Brown SE, Ausubel FM (1982) Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol 149:114–122Google Scholar
  29. Messing J (1983) New M13 vectors for cloning. Methods Enzymol 101:20–78Google Scholar
  30. Nakane PK (1968) Simultaneous localization of multiple tissue antigens using the peroxidase-labeled antibody method: a study on pituitary glands of the rat. J Histochem Cytochem 16:557–560Google Scholar
  31. Nordling M, Young S, Karlsson BG, Lundberg LG (1990) The structural gene for cytochrome c 551 from Pseudomonas aeruginosa. The nucleotide sequence shows a location downstream of the nitrite reductase gene. FEBS Lett 259:230–232Google Scholar
  32. 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 163:324–330Google Scholar
  33. Römermann D, Friedrich B (1985) Denitrification by Alcaligenes eutrophus is plasmid dependent. J Bacteriol 162:852–854Google Scholar
  34. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  35. Silvestrini MC, Galeotti CL, Gervais M, Schininà E, Barra D, Bossa F, Brunori M (1989) Nitrite reductase from Pseudomonas aeruginosa: Sequence of the gene and the protein. FEBS Lett 245:33–38Google Scholar
  36. 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 1:784–791Google Scholar
  37. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  38. Tijssen P (1985) Practice and theory of enzyme immunoassays. Elsevier, Amsterdam, pp 96–99Google Scholar
  39. Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354Google Scholar
  40. van Hartingsveldt J, Stouthamer AH (1973) Mapping and characterization of mutants of Pseudomonas aeruginosa affected in nitrate respiration in aerobic or anaerobic growth. J Gen Microbiol 74:97–106Google Scholar
  41. Viebrock A, Zumft WG (1987) Physical mapping of transposon Tn5 insertions defines a gene cluster functional in nitrous oxide respiration by Pseudomonas stutzeri. J Bacteriol 169:4577–4580Google Scholar
  42. Viebrock A, Zumft WG (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 170:4658–4668Google Scholar
  43. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the Ml3mp18 and pUC19 vectors. Gene 33:103–119Google Scholar
  44. Young RA, Davis RW (1983a) Yeast RNA polymerase II genes isolation with antibody probes. Science 222:778–782Google Scholar
  45. Young RA, Davis RW (1983b) Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 89:1194–1198Google Scholar
  46. Young RA, Bloom BR, Grosskinsky CM, Ivanyi J, Thomas D, Davis RW (1985) Dissection of Mycobacterium tuberculosis antigens using recombinant DNA. Proc Natl Acad Sci USA 82:2583–2587Google Scholar
  47. Zumft WG (1990) Molecular analysis of the denitrification system of pseudomonads. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: achievements and objectives. Chapman and Hall, New York, in pressGoogle Scholar
  48. Zumft WG, Kroneck PMH (1990) Metabolism of nitrous oxide. In: Revsbech NP, Sorensen J (eds) Denitrification in soil and sediment. FEMS Symposium Series, vol 56. Plenum, New York, pp 37–55Google Scholar
  49. Zumft WG, Matsubara T (1982) A novel kind of multi-copper protein as terminal oxidoreductase of nitrous oxide respiration in Pseudomonas perfectomarinus. FEBS Lett 148:107–112Google Scholar
  50. Zumft WG, Döhler K, Körner H (1985) Isolation and characterization of transposon Tn5-induced mutant of Pseudomonas perfectomarina defective in nitrous oxide respiration. J Bacteriol 163:918–924Google Scholar
  51. Zumft WG, Döhler K, Körner H, Löchelt S, Viebrock A, Frunzke K (1988) Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri. Arch Mikrobiol 149:492–498Google Scholar
  52. Zumft WG, Viebrock-Sambale A, Braun C (1990) Nitrous oxide reductase from denitrifying Pseudomonas stutzeri. Genes for copper-processing and properties of the deduced products, including a new member of the family of ATP/GTP-binding proteins. Eur J Biochem, 192:591–599Google Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Angelika Jüngst
    • 1
  • Cornelia Braun
    • 1
  • Walter G. Zumft
    • 1
  1. 1.Lehrstuhl für MikrobiologieUniversität KarlsruheKarlsruhe 1Germany

Personalised recommendations