Skip to main content
Log in

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

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

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.

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

Access this article

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

References

  • 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 3:235–239

    Google Scholar 

  • Bachman BJ (1983) Linkage map of Escherichia coli K12, Edition 7. Microbiol Rev 47:180–230

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res 7:1513–1523

    Google Scholar 

  • Casadaban MJ, Cohen SN (1980) Analysis of gene control signals by DNA fusion and cloning in Escherichia coli. J Mol Biol 138:179–207

    Google Scholar 

  • Chang ACY, Cohen SN (1978) Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic mini-plasmids. J Bacteriol 134:1141–1156

    Google Scholar 

  • Cole JA (1978) The rapid accumulation of large quantities of ammonia during nitrite reduction by Escherichia coli. FEMS (Microbiol Lett 4:327–329

    Google Scholar 

  • Cole JA, Ward FB (1973) Nitrite reductase-deficient mutants of Escherichia coli K12. J Gen Microbiol 76:21–29

    Google Scholar 

  • 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 85:11–22

    Google Scholar 

  • 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 120:475–483

    Google Scholar 

  • Coleman KJ, Cornish-Bowden A, Cole JA (1978) Purification and properties of nitrite reductase from Escherichia coli K12. Biochem J 175:483–493

    Google Scholar 

  • 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: 4242–4246

    Google Scholar 

  • Cossart P, Gicquel-Sanzey B (1982) Cloning and sequence of the crp gene of Escherichia coli K12. Nucleic Acids Res 10:1363–1378

    Google Scholar 

  • Hohn B, Collins J (1980) A small cosmid for efficient cloning of large DNA fragments. Gene 11:291–298

    Google Scholar 

  • Ish-Horowicz D, Burke JF (1981) Rapid and efficient cosmid cloning. Nucl Acids Res 9:2989–2998

    Google Scholar 

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

    Google Scholar 

  • 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 press

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbour Laboratory, Cold Spring Harbour, New York

    Google Scholar 

  • Marmur J (1961) A procedure for the isolation of deoxyribonucleic acid from micro-organisms. J Mol Biol 3:208–218

    Google Scholar 

  • Mejbaum-Katzenellenbogen W, Drobryszycka WH (1959) New method for quantitative determination of serum proteins separated by paper electrophoresis. Clin Chem Acta 4:515–522

    Google Scholar 

  • 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 248:2801–2814

    Google Scholar 

  • 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 106:1–12

    Google Scholar 

  • 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 128:219–222

    Google Scholar 

  • 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 248:251–264

    Google Scholar 

  • Stoker NG, Fairweather NF, Spratt BG (1982) Versatile low-copy number plasmid vectors for cloning in Escherichia coli. Gene 18:335–341

    Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Additional information

Communicated by A. Böck

Rights and permissions

Reprints and permissions

About this article

Cite this article

Macdonald, H., Cole, J. 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. Molec. Gen. Genet. 200, 328–334 (1985). https://doi.org/10.1007/BF00425444

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00425444

Keywords

Navigation