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Molecular and General Genetics MGG

, Volume 200, Issue 2, pp 328–334 | Cite as

Molecular cloning and functional analysis of the cysG and nirB genes of Escherichia coli K12, two closely-linked genes required for NADH-dependent nitrite reductase activity

  • Heather Macdonald
  • Jeff Cole
Article

Summary

We have cloned two genes, nirB+and cysG+which are required for NADH-dependent nitrite reductase to be active, from the 74 min region of the Escherichia coli chromosome. Restriction mapping and complementation analysis establish the gene order crp-nirB-cysG-aroB. Both genes are trans-dominant in merodiploids and, under some conditions, can be expressed independently. The cysG+gene can be expressed from both high and low copy number plasmids carrying a 3.6 kb PstI-EcoRI restriction fragment. Attempts to sub-clone the nirB+gene into pBR322 on a 14.5 kb EcoRI fragment were unsuccessful, but this fragment was readily sub-cloned into and expressed from the low copy number plasmid pLG338 (Stoker et al. 1982). Overproduction of the 88 kDa nitrite reductase apoprotein by strains carrying a functional nirB+gene suggests that nirB is the structural gene for this enzyme.

Keywords

Enzyme Escherichia Coli Nitrite Functional Analysis Structural Gene 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

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References

  1. Abou-Jaoudé A, Pascal M-C, Casse F, Chippaux M (1978) Isolation and phenotypes of mutants from Escherichia coli K12 defective in nitrite reductase activity. FEMS Microbiol Lett 3:235–239Google Scholar
  2. Bachman BJ (1983) Linkage map of Escherichia coli K12, Edition 7. Microbiol Rev 47:180–230Google Scholar
  3. Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523Google Scholar
  4. Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207Google Scholar
  5. Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic mini-plasmids. J Bacteriol 134:1141–1156Google Scholar
  6. Cole JA (1978) The rapid accumulation of large quantities of ammonia during nitrite reduction by Escherichia coli. FEMS (Microbiol Lett 4:327–329Google Scholar
  7. Cole JA, Ward FB (1973) Nitrite reductase-deficient mutants of Escherichia coli K12. J Gen Microbiol 76:21–29Google Scholar
  8. Cole JA, Coleman KJ, Compton BE, Kavanagh BM, Keevil CW (1974) Nitrite and ammonia assimilation by anaerobic continuous cultures of Escherichia coli. J Gen Microbiol 85:11–22Google Scholar
  9. Cole JA, Newman BM, White P (1980) Biochemical and genetic characterization of nirB mutants of Escherichia coli K12 pleiotropically defective in nitrite and sulphite reduction. J Gen Microbiol 120:475–483Google Scholar
  10. Coleman KJ, Cornish-Bowden A, Cole JA (1978) Purification and properties of nitrite reductase from Escherichia coli K12. Biochem J 175:483–493Google Scholar
  11. Collins J, Hohn B (1978) Cosmids: A type of plasmid gene-cloning vector that is packageable in vitro in bacteriophage heads. Proc Natl Acad Sci, USA, 75: 4242–4246Google Scholar
  12. Cossart P, Gicquel-Sanzey B (1982) Cloning and sequence of the crp gene of Escherichia coli K12. Nucleic Acids Res 10:1363–1378Google Scholar
  13. Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298Google Scholar
  14. Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucl Acids Res 9:2989–2998Google Scholar
  15. Jackson RH, Cornish-Bowden A, Cole JA (1981) Prosthetic groups of the NADH-dependent nitrite reductase from Escherichia coli K12. Biochem J 193:861–867Google Scholar
  16. Macdonald H, Pope NR, Cole JA (1985) Isolation, characterization and complementation analysis of nirB mutants of Escherichia coli deficient only in NADH-dependent nitrite reductase activity. J Gen Microbiol, in pressGoogle Scholar
  17. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbour Laboratory, Cold Spring Harbour, New YorkGoogle Scholar
  18. Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218Google Scholar
  19. Mejbaum-Katzenellenbogen W, Drobryszycka WH (1959) New method for quantitative determination of serum proteins separated by paper electrophoresis. Clin Chem Acta 4:515–522Google Scholar
  20. Murphy WJ, Siegel LM, Kamin H, Rosenthal D (1973) Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria II. Identification of a new class of heme prosthetic group: an iron-tetrahydroporphyrin (isobacteriochlorin type) with eight carboxylic acid groups. J Biol Chem 248:2801–2814Google Scholar
  21. Newman BM, Cole JA (1978) The chromosomal location and pleiotropic effects of mutations in the nirA +gene of Escherichia coli K12: the essential role of nirA +in nitrite reduction and in other anaerobic redox reactions. J Gen Microbiol 106:1–12Google Scholar
  22. Pope NR, Cole JA (1982) Generation of a membrane potential by one of two independent pathways for nitrite reduction by Escherichia coli. J Gen Microbiol 128:219–222Google Scholar
  23. Siegel LM, Murphy MJ, Kamin H (1973) Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of Enterobacteria I. The Escherichia coli hemoflavoprotein: molecular parameters and prosthetic groups. J Biol Chem 248:251–264Google Scholar
  24. Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low-copy number plasmid vectors for cloning in Escherichia coli. Gene 18:335–341Google Scholar

Copyright information

© Springer-Verlag 1985

Authors and Affiliations

  • Heather Macdonald
    • 1
  • Jeff Cole
    • 1
  1. 1.Department of BiochemistryUniversity of BirminghamBirminghamU.K.

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