Skip to main content
SpringerLink
Log in

Cloning and organisation of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae

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

Summary

By DNA hybridisation, restriction fragments of genomic DNA from Azotobacter chroococcum and A. vinelandii bearing sequences homologous to Klebsiella pneumoniae nitrogenase structural genes were detected. These were different in the two species and inconsistent with the arrangement of the homologous sequences as a contiguous cluster of unique genes. The use of a DNA probe specific for nifH showed that in A. chroococcum two nifH-like sequences were present in the genome. From gene libraries for A. chroococcum, several recombinant cosmid clones bearing nif genes were identified and physically mapped. One copy of the nifH-like sequences was closely linked to nifD and K, the order of genes being as for K. pneumoniae. This cluster was sub-cloned into the broad host-range vector pKT230. The resultant plasmid complemented for C2H2-reduction but not growth in N2 several Nif- mutants of A. vinelandii and K. pneumoniae and also abolished growth in N2 in Nif+ parents. The inhibition was ascribed to a short region adjacent to nifH, which probably corresponds to the promoter as its inhibitory affects were alleviated by provision of K. pneumoniae nifA in multiple copies. 3 sizes of transcripts are produced from the region containing nifH and nifD of A. chroococcum in cultures derepressing for nif. A region bearing homology to a fragment of the K. pneumoniae nif cluster bearing nifV was identified 15 Kb away from nifHDK in A. chroococcum however the order of genes is probably similar to that of K. pneumoniae.

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

References

  • Bagdasarian M, Franklin FCH, Lurz R, Rückert B, bagdasarian MM, Timmis KN (1981) Specific purpose cloning vectors. II. Broad host range, high copy number RSF1010-derived vectors, and a host: vector system for gene cloning in Pseudomonas. Gene 16:237–247

    Google Scholar 

  • Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1513–1525

    Google Scholar 

  • Bishop PE, Bott KF (1983) Molecular cloning of nif DNA from Azotobacter vinelandii. Abstr Ann Meeting AmSoc Microbiol:188

  • Bishop PF, Brill WJ (1977) Genetic analysis of Azotobacter vinelandii mutant strains unable to fix nitrogen. J Bacteriol 130:954–956

    Google Scholar 

  • Bishop PE, Jarlenski DML, Hetherington DR (1980) Evidence for an alternative nitrogen fixation system in Azotobacter vinelandii. Proc Natl Acad Sci USA 77:7342–7346

    Google Scholar 

  • Bishop PE, Jarlenski DML, Hetherington DR (1982) Expression of an alternative nitrogen fixation system in Azotobacter vinelandii. J Bacteriol 150:1244–1251

    Google Scholar 

  • Boyer HB, 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 

  • Buchanan-Wollaston V, Cannon MC, Beynon JL, Cannon FC (1981a) Role of the nifA gene product in the regulation of nif expression in Klebsiella pneumoniae. Nature 294:776–778

    Google Scholar 

  • Buchanan-Wollaston V, Cannon MC, Cannon FC (1981b) The use of cloned nif (nitrogen fixation) DNA to investigate transcriptional regulation of nif expression in Klebsiella pneumoniae. Mol Gen Genet 184:102–106

    Google Scholar 

  • Bush JA, Wilson PW (1959) A non-gummy chromogenic strain of Azotobacter vinelandii. Nature 184:381–382

    Google Scholar 

  • Cannon FC, Dixon RA, Postgate JR, Primrose SB (1974) Chromosomal integration of Klebsiella nitrogen fixation genes in Escherichia coli. J Gen Microbiol 80:227–239

    Google Scholar 

  • Cannon FC, Riedel GE, Ausubel FM (1979) Overlapping sequences of Klebsiella pneumoniae nif DNA cloned and characterised. Mol Gen Genet 174:59–66

    Google Scholar 

  • Denhardt DT (1966) A membrane-filter technique for the detection of complementary DNA. Biochem Biophys Res Commun 23:641–646

    Google Scholar 

  • Dixon R, Kennedy C, Kondorosi A, Krishnapillai V, Merrick M (1977) Complementation analysis of Klebsiella pneumoniae mutants defective in nitrogen fixation. Mol Gen Genet 157:189–198

    Google Scholar 

  • Dixon RA, Eady R, Espin G, Hill S, Iaccarino M, Kahn D, Merrick M (1980) Analysis of the regulation of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster with gene fusions. Nature 286:128–132

    Google Scholar 

  • Figurski D, Helinski D (1979) Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc Natl Acad Sci USA 76:1648–1652

    Google Scholar 

  • Grosveld FG, Dahl H-HM, de Boer E, Flavell RA (1981) Isolation of β-globin-related genes from a human cosmid library. Gene 13:227–237

    Google Scholar 

  • Grosveld FG, Lund T, Murray EJ, Mellor AL, Dahl H-HM, Flavell RA (1982) The construction of cosmid libraries which can by used to transform eukaryotic cells. Nucl Acids Res 10:6715–6732

    Google Scholar 

  • Hanahan D, Meselson M (1980) Plasmid screening at high colony density. Gene 10:63–67

    Google Scholar 

  • Hausinger RP, Howard JB (1982) The amino acid sequence of the nitrogenase iron protein from Azotobacter vinelandii. J Biol Chem 257:2483–2490

    Google Scholar 

  • Hill S, Kavanagh EP (1980) Roles of nifF and nifJ gene products in electron transpor to nitrogenase in Klebsiella pneumoniae. J Bacteriol 141:470–475

    Google Scholar 

  • Hill S, Kennedy C, Kavanagh E, Goldberg R, Hanau R (1981) Nitrogen fixation gene (nifL) involved in oxygen regulation of nitrogenase synthesis in K. pneumoniae. Nature 290:424–426

    Google Scholar 

  • Hohn B (1979) In vitro packaging of λ and cosmid DNA. In: Wu R (ed) Recombinant DNA. Methods in Enzymology, vol 68. Academic Press, New York, pp 299–309

    Google Scholar 

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

    Google Scholar 

  • Kallas T, Rebiere MC, Rippka R, Tandeau de Marsac N (1983) The structural nif gene of the Cyanobacteria Gleothece sp and Calothrix sp. share homology with those of Anabaena sp, but the Gleothece genes have a different arrangement. J Bacteriol 144:427–431

    Google Scholar 

  • Kennedy C (1977) Linkage map of the nitrogen fixation (nif) genes in Klebsiella pneumoniae. Mol Gen Genet 157:199–204

    Google Scholar 

  • Kennedy C, Robson RL (1983) Activation of nif gene expression in Azotobacter by the nifA gene product of Klebsiella pneumoniae. Nature 301:626–628

    Google Scholar 

  • Krol AJM, Hontelez JGJ, Roozendaal B, van Kammen A (1982) On the operon structure of the nitrogenase genes of Rhizobium leguminosarum and Azotobacter vinelandii. Nucl Acids Res 10:4147–4157

    Google Scholar 

  • Kushner SR (1978) An improved method for transformation of Escherichia coli with ColEl derived plasmids. In: Boyer HW and Nicosia S (eds) Genetic engineering. Elsevier/North-Holland Biomedical Press, Amsterdam, p 17–23

    Google Scholar 

  • MacNeil T, MacNeil D, Roberts GP, Supiano MA, Brill WJ (1978) Fine structure mapping and complementation analysis of nif (nitrogen fixation) genes inKlebsiella pneumoniae. J Bacteriol 136:253–266

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY

    Google Scholar 

  • Mclean PA, Dixon RA (1981) Requirement of nifV gene for production of wild-type nitrogenase enzyme in Klebsiella pneumoniae. Nature 292:655–656

    Google Scholar 

  • Merrick M, Filser M, Dixon R, Elmerich C, Sibold L, Houmard J (1980) Use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster. J Gen Microbiol 117:509–520

    Google Scholar 

  • Merrick M, Hill S, Hennecke H, Hahn M, Dixon R, Kennedy C (1982) Repressor properties of the nifL gene product in Klebsiella pneumoniae. Mol Gen Genet 185:75–81

    Google Scholar 

  • Meyerowitz EM, Guild GM, Prestidge LS, Hogness DS (1980) A new cosmid vector and its uses. Gene 11:271–282

    Google Scholar 

  • Murray NE, Brammar WJ, Murray K (1977) Lamboid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet 150:53–61

    Google Scholar 

  • Nieva-Gomez D, Roberts GP, Klevickis S, Bill WJ (1980) Electron transport to nitrogenase in Klebsiella pneumoniae. Proc Natl Acad Sci USA 77:2555–2558

    Google Scholar 

  • Premakumar R, Lemos EM, Bishop PE (1984) Evidence for two dinitrogenase reductases under regulatory control by molybdenum in Azotobacter vinelandii. Biochim Biophys Acta 797:64–71

    Google Scholar 

  • Pühler A, Klipp W (1981) Fine structure analysis of the gene region of N2-fixation (nif) of Klebsiella pneumoniae. In: Bothe H, Trebst A (eds) Biology of inorganic nitrogen and sulphur. Springer-Verlag, Berlin Heidelberg New York, pp 276–286

    Google Scholar 

  • Quinto C, de la Vega H, Flores M, Fernandez L, Ballado T, Soberon G, Palacios R (1982) Reiteration of nitrogen fixation gene sequences in Rhizobium phaseoli. Nature 299:727–726

    Google Scholar 

  • Rice D, Mazur BJ, Haselkorn R (1982) Isolation and physical mapping of nitrogen fixation gene from the Cyanobacterium Anabaena 7120. J Biol Chem 257:13157–13163

    Google Scholar 

  • Riedel GE, Ansubel FM, Cannon FC (1979) Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae. Proc Natl Acad Sci USA 76:2866–2870

    Google Scholar 

  • Riedel GE, Brown SE, Ausubel FM (1983) Nitrogen fixation by Klebsiella pneumoniae is inhibited by certain multicopy hybrid nif plasmids. J Bacteriol 153:45–56

    Google Scholar 

  • Rigby PWJ, Dieckmann M, Rhodes C, Berg P (1977) Labelling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol 113:237–251

    Google Scholar 

  • Rizzo M, Bishop PE (1983) Physical mapping of recombinant plasmids containing nif DNA from Azotobacter vinelandii. Abstr Ann Meeting Am Soc Microbiol: 188

  • Roberts GP, Brill WJ (1980) Gene-product relationship of the nif regulon of Klebsiella pneumoniae. J Bacteriol 144:210–216

    Google Scholar 

  • Roberts GP, MacNeil T, MacNeil D, Brill WJ (1978) Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol 136:267–279

    Google Scholar 

  • Robson RL, Postgate JR (1980) Oxygen and hydrogen in biological nitrogen fixation. Annu Rev Microbiol 34:183–207

    Google Scholar 

  • Robson RL, Kennedy CK, Postgate JR (1983) Progress in comparative genetics of nitrogen fixation. Canad J Microbiol 29:954–966

    Google Scholar 

  • Robson RL, Chesshyre J, Wheeler C, Jones R, Woodley P, Postgate J (1984) Genome size and complexity in Azotobacter chroococcum. J Gen Microbiol 130:1603–1612

    Google Scholar 

  • Ruvkun GB, Ausubel F (1980) Interspecies homology of nitrogenase genes. Proc Natl Acad Sci USA 77:191–195

    Google Scholar 

  • Scolnik PA, Haselkorn R (1984) Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata. Nature 307:289–292

    Google Scholar 

  • Shah VK, Brill WJ (1977) Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci USA 74:3249–3253

    Google Scholar 

  • Shah VK, Davis LC, Gordon JK, Orme-Johnson WH, Brill WJ (1973) Nitrogenase III. Nitrogenaseless mutants of Azotobacter vinelandii: activities, cross-reactions, and EPR spectra. Biochim Biophys Acta 292:246–255

    Google Scholar 

  • Shah VK, Stacey G, Brill WJ (1983) Electron transport to Nitrogenase. Purification and characterisation of pyruvate: flavodoxin oxidoreductase, the nifJ gene product. J Biol Chem 258:12064–12068

    Google Scholar 

  • Streicher SL, Shanmugam KT, Ausubel F, Morandi C, Goldberg RB (1974) Regulation of nitrogen fixation in Klebsiella pneumoniae: evidence for a role of glutamine synthetase as a regulator of nitrogenase synthesis. J Bacteriol 120:815–821

    Google Scholar 

  • Thorneley RNF, Lowe DJ (1983) Nitrogenase of Klebsiella pneumoniae. Kinetics of the dissociation of oxidised iron protein from molybdenum-iron protein: Identification of the rate-limiting step for substrate reduction. Biochem J 215:393–403

    Google Scholar 

  • Thuring RWJ, Sanders JPM, Borst P (1975) A freeze-squeeze method for recovering long DNA from agarose gels. Anal Biochem 66:213–220

    Google Scholar 

  • Wahl GM, Stern M, Stark GR (1979) Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethylpaper and rapid hybridisation by using dextran sulphate. Proc Natl Acad Sci USA 76:3683–3687

    Google Scholar 

  • Wieslander L (1979) A simple method to recover intact high molecular weight RNA and DNA after electrophoretic separation in low gelling temperature agarose gels. Anal Biochem 98:305–309

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. AFRC, Unit of Nitrogen Fixation, University of Sussex, BN1 9RQ, Brighton, UK

    Robert Jones, Paul Woodley & Robert Robson

Authors
  1. Robert Jones
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Paul Woodley
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Robert Robson
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by J. Schell

Rights and permissions

Reprints and permissions

About this article

Cite this article

Jones, R., Woodley, P. & Robson, R. Cloning and organisation of some genes for nitrogen fixation from Azotobacter chroococcum and their expression in Klebsiella pneumoniae . Mol Gen Genet 197, 318–327 (1984). https://doi.org/10.1007/BF00330980

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00330980

Keywords

Search

Navigation