Genetics of Nitrogen Fixation in Free-Living Organisms

  • A. Pühler
  • W. Klipp


Using the acetylene-reduction assay, a long list of organisms has been prepared that are able to fix nitrogen under free-living conditions (Table 1). This list contains anaerobic, microaerobic, facultative and aerobic microorganisms. It is worth noting that all these microorganisms are of procaryotic origin and that no eucaryotic organism with the capacity to fix nitrogen is known. Although a lot is known about the physiology and biochemistry of free-living, nitrogen-fixing organisms, only very few have been used for genetic studies of the nitrogen-fixation process. The facultative microorganism Klebsiella pneumoniae has been mainly used to elucidate the genetic background of biological nitrogen fixation and is, therefore, extensively discussed in this paper. Besides Klebsiella, the genetics of free-living, nitrogen-fixing bacteria is still very virginal. For Azotobacter vinelandii, nitrogenase-defective mutants have been reported.1-3 Using the Azotobacter transformation system, Bishop and Brill4 were able to locate the mutations as a cluster on the chromosome. Mutants defective in nitrogen fixation (nif -) were also isolated for cyanobacteria,5-7 but, due to the lack of a gene-transfer system, no further genetic studies could be performed with these mutants.


Nitrogen Fixation Klebsiella Pneumoniae EcoRI Fragment HindIII Fragment Operon Structure 
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  1. 1.
    R. J. Fisher and W. J. Brill, Mutants of Azobotacter vinelandii Unable to Fix Nitrogen, Biochem. Biophys. Acta 184:99 (1969).PubMedCrossRefGoogle Scholar
  2. 2.
    G. J. Sorger and D. Trofimenkoff, Nitrogenaseless Mutants of Azotobacter vinelandii, Proc. Natl. Acad, Sci. USA 65:74 (1970).CrossRefGoogle Scholar
  3. 3.
    V. K. Shah, L. C. Davis, J. K. Gordon, W. H. Orme-Johnson, and W. J. Brill, Nitrogenase III. Nitrogenaseless Mutants of Azotobacter vinelandii: Activities, Cross Reactions and EPR Spectra, Biochim. Biophys. Acta 292:246 (1973).PubMedCrossRefGoogle Scholar
  4. 4.
    P. E. Bishop and W. J. Brill, Genetic Analysis of Azotobactervinelandii Mutant Strains Unable to Fix Nitrogen, J. Bacteriol. 130:954 (1977).PubMedGoogle Scholar
  5. 5.
    C. van Baalen, Mutagenesis and Genetic Recombination, in: “The Biology of Blue-Green Algae,” N. G. Carr and B. A. Whitton, eds., Blackwell Scientific Publications, Oxford, p. 201 (1973).Google Scholar
  6. 6.
    T. C. Currier, J. F. Haury, and C. P. Wolk, Isolation and Preliminary Characterization of Auxotrophs of a Filamentous Cyanobacterium, J. Bacteriol. 129:1556 (1977).PubMedGoogle Scholar
  7. 7.
    J. F. Haury and C. P. Wolk, Classes of Anabaena variabilis Mutants with Oxygen Sensitive Nitrogenase Activity, J. Bacteriol. 136:688 (1978).PubMedGoogle Scholar
  8. 8.
    M. Solioz and B. Marrs, The Gene Transfer Agent of Rhodopseudomonas capsulata: Purification and Characterization of its Nucleic Acids, Arch. Biochem. Biophys. 181:300 (1977).PubMedCrossRefGoogle Scholar
  9. 9.
    J. D. Wall, P. F. Weaver, and H. Gest, Genetic Transfer of Nitrogenase-Hydrogenase Activity in Rhodopseudomonas capsulata, Nature 258:630 (1975).PubMedCrossRefGoogle Scholar
  10. 10.
    W. R. Sistrom, Transfer of Chromosomal Genes Mediated by Plasmid R68.45 in Rhodopseudomonas sphaeroides, J. Bacteriol. 131:526 (1977).PubMedGoogle Scholar
  11. 11.
    L. Miller and S. Kaplan, Plasmid Transfer and Expression in Rhodopseudomonas sphaeroides, Arch. Biochem. Biophys. 187:229 (1978).PubMedCrossRefGoogle Scholar
  12. 12.
    M. A. Simon and W. J. Brill, Mutant of Clostridium pasteurianum That Does Not Fix Nitrogen, J. Bacteriol. 105:65 (1971).PubMedGoogle Scholar
  13. 13.
    S. Streicher, E. Gurney, and R. C. Valentine, Transduction of the Nitrogen-Fixation Genes in Klebsiella pneumoniae, Proc. Natl. Acad. Sci. USA 68:1174 (1971).PubMedCrossRefGoogle Scholar
  14. 14.
    R. A. Dixon and J. R. Postgate, Transfer of Nitrogen Fixation Genes by Conjugation in Klebsiella pneumoniae, Nature 234:47 (1971).PubMedCrossRefGoogle Scholar
  15. 15.
    S. L. Streicher, E. Gurney, and R. C. Valentine, The Nitrogen Fixation Gene, Nature 239:495 (1972).PubMedCrossRefGoogle Scholar
  16. 16.
    K. T. Shanmugam, A. S. Loo, and R. C. Valentine, Deletion Mutants of Nitrogen Fixation in Klebsiella pneumoniae : Mapping of a Cluster of nif Genes Essential for Nitrogenase Activity, Biochim. Biophys. Acta 338:545 (1974).CrossRefGoogle Scholar
  17. 17.
    R. N. Rao, Mutational Alteration of a Nitrogen-Fixing Bacterium to Sensitivity to Infection by Bacteriophage Mu: Isolation of nif Mutations of Klebsiella pneumoniae M5al Induced by Mu, J. Bacteriol. 128:356 (1976).PubMedGoogle Scholar
  18. 18.
    R. A. Dixon and F. C. Cannon, Recent Advances in the Genetics of Nitrogen Fixation, in: “Symbiotic Nitrogen Fixation in Plants,” IBP 7, P. S. Nutman, ed., Cambridge University Press, London, p. 3 (1976).Google Scholar
  19. 19.
    D. MacNeil, M. A. Supiano, and W. J. Brill, Order of Genes Near nif in Klebsiella pneumoniae, J. Bacteriol. 138:1041 (1979).PubMedGoogle Scholar
  20. 20.
    R. T. St. John, H. M. Johnston, C. Seidman, D. Garfinkel, J. K. Gordon, V. K. Shah, and W. J. Brill, Biochemistry and Genetics of Klebsiella pneumoniae Mutant Strains Unable to Fix N2, J. Bacteriol. 121:759 (1975).Google Scholar
  21. 21.
    C. Kennedy, Linkage Map of the Nitrogen (nif) Genes in Klebsiella pneumoniae, Molec. Gen. Genet. 157:199 (1977).PubMedCrossRefGoogle Scholar
  22. 22.
    R. A. Dixon and J. R. Postgate, Genetic Transfer of Nitrogen Fixation from Klebsiella pneumoniae to Escherichia coli, Nature 237:102 (1972).PubMedCrossRefGoogle Scholar
  23. 23.
    R. A. Dixon, Construction of an F -Factor and Derivative Plasmids Carrying the nif Genes from Klebsiella pneumoniae, Heredity 33:134 (1974).Google Scholar
  24. 24.
    R. A. Dixon, F. C. Cannon, and A. Kondorosi, Construction of a P Plasmid Carrying Nitrogen Fixation Genes from Klebsiella pneumoniae, Nature 260:268 (1976).PubMedCrossRefGoogle Scholar
  25. 25.
    F. C. Cannon, R. A. Dixon, and J. R. Postgate, Derivation and Properties of F-Prime Factors in Escherichia coli Carrying Nitrogen Fixation Genes from Klebsiella pneumoniae, J. Gen. Microbiol. 93:111 (1976).PubMedGoogle Scholar
  26. 26.
    F. C. Cannon and J. R. Postgate, Expression of Klebsiella Nitrogen Fixation Genes (nif) in Azotobacter, Nature 260:271 (1976).PubMedCrossRefGoogle Scholar
  27. 27.
    J. R. Postgate and V. Krishnapillai, Expression of Klebsiellanif and his Genes in Salmonella typhimurium, J. Gen. Microbiol. 98:379 (1977).PubMedGoogle Scholar
  28. 28.
    M. M. K. Filser, Genetic Analysis of the Klebsiella pneumoniae Nitrogen Fixation Gene Cluster: Plasmid Construction and Transposon Mutagenesis, D. Phil. Thesis, University of Sussex (1980).Google Scholar
  29. 29.
    R. Dixon, C. Kennedy, A. Kondorosi, V. Krishnapillai, and M. Merrick, Complementation Analysis of Klebsiella pneumoniae Mutants Defective in Nitrogen Fixation, Molec. Gen. Genet. 157:189 (1977).PubMedCrossRefGoogle Scholar
  30. 30.
    T. MacNeil, W. J. Brill, and M. M. Howe, Bacteriophage Mu-Induced Deletions in a Plasmid Containing the nif (N2-Fixation) Genes of Klebsiella pneumoniae, J. Bacteriol. 134:821 (1978).PubMedGoogle Scholar
  31. 31.
    M. Merrick, M. Filser, C. Kennedy, and R. Dixon, Polarity of Mutations Induced by Insertion of Transposons Tn5, Tn7, and TnlO into the nif Gene Cluster of Klebsiella pneumoniae, Molec. Gen. Genet. 165:103 (1978).PubMedCrossRefGoogle Scholar
  32. 32.
    C. Elmer ich, J. Houmard, L. Sibold, I. Manheimer, and N. Charpin, Genetic and Biochemical Analysis of Mutants Induced by Bacteriophage Mu DNA Integration into Klebsiella pneumoniae Nitrogen Fixation Genes, Molec. Gen. Genet. 165:181 (1978).CrossRefGoogle Scholar
  33. 33.
    M. Merrick, M. Filser, R. Dixon, S. Eimerich, L. Sibold, and J. Houmard, 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 (1980).PubMedGoogle Scholar
  34. 34.
    G. P. Roberts, T. MacNeil, D. MacNeil, and W. J. Brill, Regulation and Characterization of Protein Products Coded by the nif (Nitrogen Fixation) Genes of Klebsiella pneumoniae, J. Bacteriol. 136:267 (1978).PubMedGoogle Scholar
  35. 35.
    W. K. Shah and W. J. Brill, Isolation of an Iron-Molybdenum Co-Factor from Nitrogenase, Proc. Natl. Acad. Sci. USA 74:3249 (1977).PubMedCrossRefGoogle Scholar
  36. 36.
    C. Kennedy, F. Cannon, M. Cannon, R. Dixon, S. Hill, J. Jensen, S. Kumar, P. McLean, M. Merrick, R. Robson, and J. Postgate, Recent Advances in the Genetics and Regulation of Nitrogen Fixation, in: “Current Perspectives in Nitrogen Fixation,” A. H. Gibson and W. E. Newton, eds., Australian Academy of Science, Canberra, p. 146 (1981).Google Scholar
  37. 37.
    J. M. Leonardo and R. B. Goldberg, Regulation of Nitrogen Metabolism in Glutamine Auxotrophs of Klebsiella pneumoniae, J. Bacteriol. 142:99 (1980).PubMedGoogle Scholar
  38. 38.
    J. Houmard, D. Bogusz, R. Bigault, and C. Elmerich, Characterization and Kinetics of the Biosynthesis of Some Nitrogen Fixation (nif) Gene Products in Klebsiella pneumoniae, Biochimie, in press.Google Scholar
  39. 39.
    R. Dixon, R. Eady, G. Espin, S. Hill, M. Iaccarino, D. Kahn, and M. Merrick, Analysis of Regulation of the Klebsiella pneumoniae Nitrogen Fixation (nif) Gene Cluster with Gene Fusions, Nature 286:128 (1980).PubMedCrossRefGoogle Scholar
  40. 40.
    R. Dixon, C. Kennedy, and M. Merrick, Genetic Control of Nitrogen Fixation, in: “Genetics as a Tool in Microbiology,” S. W. Glover and D. A. Hopwood, eds., Cambridge University Press, p. 161 (1981).Google Scholar
  41. 41.
    F. M. Ausubel, G. Reidel, F. Cannon, A. Peskin, and R. Margolskee, Cloning Nitrogen Fixation Genes from Klebsiella pneumoniae in vitro and the Isolation of nif Promoter Mutants Affecting Glutamine Synthetase Regulation, in: “Genetic Engineering for Nitrogen Fixation,” A. Hollaender, ed., Plenum Press, New York, p. III (1977).Google Scholar
  42. 42.
    F. C. Cannon, G. E. Reidel, and F. M. Ausubel, A Recombinant Plasmid which Carries Part of the Nitrogen Fixation (nif) Gene Cluster of Klebsiella pneumoniae, Proc. Natl. Acad. Sci. USA 74:2963 (1977).PubMedCrossRefGoogle Scholar
  43. 43.
    A. Pühler, H. J. Burkhardt, and W. Klipp, Cloning of the Entire Region for Nitrogen Fixation from Klebsiella pneumoniae on a Multicopy Plasmid Vehicle in E. coli, Molec. Gen. Genet. 176:17 (1979).PubMedCrossRefGoogle Scholar
  44. 44.
    A. Pühler and W. Klipp, Fine Structure Analysis of the Gene Region for N2-Fixation (nif) of Klebsiella pneumoniae, in: “Biology of Inorganic Nitrogen and Sulfur,” H. Bothe and A. Trebst, eds., Springer-Verlag, Berlin-Heidelberg-New York, p. 275 (1981).Google Scholar
  45. 45.
    A. Pühler, H. J. Burkhardt, F. C. Cannon, and W. Wohlleben, Spontaneous Degradation of pRDl DNA into Unique Size Classes is recA Dependent, Molec. Gen. Genet. 171:1 (1979).PubMedCrossRefGoogle Scholar
  46. 46.
    A. Pühler, H. J. Burkhardt, and W. Klipp, Cloning in Escherichia coli the Genomic Region of Klebsiella pneumoniae which Encodes Genes Responsible for Nitrogen Fixation, in: “Plasmids of Medical, Environmental and Commercial Importance,” K. Timmis and A. Pühler, eds., Elsevier/North-Holland Biomedical Press, Amsterdam-New York, p. 435 (1979).Google Scholar
  47. 47.
    W. Goebel, W. Lindenmaier, F. Pfeiffer, H. Schrempf, B. Schelle, Transposition and Insertion of Intact, Deleted and Enlarged Ampicillin Transposon Tn3 from MiniRl (Rsc) Plasmids into Transfer Factors, Molec. Gen. Genet. 157:119 (1977).PubMedCrossRefGoogle Scholar
  48. 48.
    A. C. Y. Chang and S. N. Cohen, Construction and Characterization of Amplifiable Multicopy DNA Cloning Vehicles Derived from the P15A Cryptic Miniplasmid, J. Bacteriol. 134:1141 (1978).PubMedGoogle Scholar
  49. 49.
    F. C. Cannon, G. E. Reidel, and F. M. Ausubel, Overlapping Sequences of Klebsiella pneumoniae nif DNA Cloned and Characterized, Molec. Gen. Genet. 174:59 (1979).PubMedCrossRefGoogle Scholar
  50. 50.
    F. J. Le Grice and H. Matzura, Localization of the Transscription Initiation Site of the Chloramphenicol Resistance Gene on Plasmid pAC184, FEBS Lett. 113:42 (1980).PubMedCrossRefGoogle Scholar
  51. 51.
    M. P. Nuti, A. A. Lepidi, R. K. Prakash, R. A. Schilperoort, and F. C. Cannon, Evidence for Nitrogen Fixation (nif) Genes on Indigenous Rhizobium Plasmids, Nature 282:533 (1979).CrossRefGoogle Scholar
  52. 52.
    B. Mazur, D. Rice, and R. Haselkorn, Identification of Blue-Green Algal Nitrogen Fixation Genes by Using Heterologous DNA Hybridization Probes, Proc. Natl. Acad. Sci. USA 77:186 (1980).PubMedCrossRefGoogle Scholar
  53. 53.
    G. B. Ruvkun and F. M. Ausubel, Interspecies Homology of Nitrogenase Genes, Proc. Natl. Acad. Sci. USA 77:191 (1980).PubMedCrossRefGoogle Scholar
  54. 54.
    J. Postgate, Foreward, in: “Recent Advances in Biological Nitrogen Fixation,” N. S. Subba Rao, ed., Edward Arnold (Publishers) Limited, London, p. V. (1980).Google Scholar

Copyright information

© Plenum Press, New York 1983

Authors and Affiliations

  • A. Pühler
    • 1
  • W. Klipp
    • 1
  1. 1.Lehrstuhl fur GenetikUniversität BielefeldBielefeld 1West Germany

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