Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1991 Jan;173(1):365–371. doi: 10.1128/jb.173.1.365-371.1991

Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria.

E Fallik 1, Y K Chan 1, R L Robson 1
PMCID: PMC207195  PMID: 1987127

Abstract

Strains of aerobic, microaerobic, nonsymbiotic, and symbiotic dinitrogen-fixing bacteria were screened for the presence of alternative nitrogenase (N2ase) genes by DNA hybridization between genomic DNA and DNA encoding structural genes for components 1 of three different enzymes. A nifDK gene probe was used as a control to test for the presence of the commonly occurring Mo-Fe N2ase, a vnfDGK gene probe was used to show the presence of V-Fe N2ase, and an anfDGK probe was used to detect Fe N2ase. Hitherto, all three enzymes have been identified in Azotobacter vinelandii OP, and all but the Fe N2ase are present in Azotobacter chroococcum ATCC 4412 (MCD1). Mo-Fe N2ase and V-Fe N2ase structural genes only were confirmed in this strain and in two other strains of A. chroococcum (ATCC 480 and ATCC 9043). A similar pattern was observed with Azotobacter beijerinckii ATCC 19360 and Azotobacter nigricans ATCC 35009. Genes for all three systems are apparently present in two strains of Azotobacter paspali (ATCC 23367 and ATCC 23833) and also in Azomonas agilis ATCC 7494. There was no good evidence for the existence of any genes other than Mo-Fe N2ase structural genes in several Rhizobium meliloti strains, cowpea Rhizobium strain 32H1, or Bradyrhizobium japonicum. Nitrogenase and nitrogenase genes in Azorhizobium caulinodans behaved in an intermediate fashion, showing (i) the formation of ethane from acetylene under Mo starvation, a characteristic of alternative nitrogenases, and (ii) a surprising degree of cross-hybridization to the vnfDGK, but not the anfDGK, probe. vnfDGK- and anfDGK-like sequences were not detected in two saccharolytic Pseudomonas species or Azospirillum brasilense Sp7. The occurrence of alternative N2ases seems restricted to members of the family Azotobacteraceae among the aerobic and microaerobic diazotrophs tested, suggesting that an ability to cope with O2 when fixing N2 may be an important factor influencing the distribution of alternative nitrogenases.

Full text

PDF
366

Images in this article

367Image
on p.367
369Image
on p.369

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Barraquio W. L., Dumont A., Knowles R. Enumeration of free-living aerobic n(2)-fixing h(2)-oxidizing bacteria by using a heterotrophic semisolid medium and most-probable-number technique. Appl Environ Microbiol. 1988 Jun;54(6):1313–1317. doi: 10.1128/aem.54.6.1313-1317.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Birnboim H. C., Doly J. A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucleic Acids Res. 1979 Nov 24;7(6):1513–1523. doi: 10.1093/nar/7.6.1513. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Brigle K. E., Newton W. E., Dean D. R. Complete nucleotide sequence of the Azotobacter vinelandii nitrogenase structural gene cluster. Gene. 1985;37(1-3):37–44. doi: 10.1016/0378-1119(85)90255-0. [DOI] [PubMed] [Google Scholar]
  4. Chisnell J. R., Premakumar R., Bishop P. E. Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii. J Bacteriol. 1988 Jan;170(1):27–33. doi: 10.1128/jb.170.1.27-33.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Donald R. G., Nees D. W., Raymond C. K., Loroch A. I., Ludwig R. A. Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes. J Bacteriol. 1986 Jan;165(1):72–81. doi: 10.1128/jb.165.1.72-81.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Eady R. R., Robson R. L., Richardson T. H., Miller R. W., Hawkins M. The vanadium nitrogenase of Azotobacter chroococcum. Purification and properties of the VFe protein. Biochem J. 1987 May 15;244(1):197–207. doi: 10.1042/bj2440197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Haahtela K., Helander I., Nurmiaho-Lassila E. L., Sundman V. Morphological and physiological characteristics and lipopolysaccharide composition of N2-fixing (C2H2-reducing) root-associated Pseudomonas sp. Can J Microbiol. 1983 Aug;29(8):874–880. doi: 10.1139/m83-142. [DOI] [PubMed] [Google Scholar]
  8. Hales B. J., Case E. E., Morningstar J. E., Dzeda M. F., Mauterer L. A. Isolation of a new vanadium-containing nitrogenase from Azotobacter vinelandii. Biochemistry. 1986 Nov 18;25(23):7251–7255. doi: 10.1021/bi00371a001. [DOI] [PubMed] [Google Scholar]
  9. Hales B. J., Langosch D. J., Case E. E. Isolation and characterization of a second nitrogenase Fe-protein from Azotobacter vinelandii. J Biol Chem. 1986 Nov 15;261(32):15301–15306. [PubMed] [Google Scholar]
  10. Joerger R. D., Jacobson M. R., Premakumar R., Wolfinger E. D., Bishop P. E. Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii. J Bacteriol. 1989 Feb;171(2):1075–1086. doi: 10.1128/jb.171.2.1075-1086.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Joerger R. D., Loveless T. M., Pau R. N., Mitchenall L. A., Simon B. H., Bishop P. E. Nucleotide sequences and mutational analysis of the structural genes for nitrogenase 2 of Azotobacter vinelandii. J Bacteriol. 1990 Jun;172(6):3400–3408. doi: 10.1128/jb.172.6.3400-3408.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. 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. 1984;197(2):318–327. doi: 10.1007/BF00330980. [DOI] [PubMed] [Google Scholar]
  13. Pau R. N., Mitchenall L. A., Robson R. L. Genetic evidence for an Azotobacter vinelandii nitrogenase lacking molybdenum and vanadium. J Bacteriol. 1989 Jan;171(1):124–129. doi: 10.1128/jb.171.1.124-129.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Quiviger B., Franche C., Lutfalla G., Rice D., Haselkorn R., Elmerich C. Cloning of a nitrogen fixation (nif) gene cluster of Azospirillum brasilense. Biochimie. 1982 Jul;64(7):495–502. doi: 10.1016/s0300-9084(82)80165-x. [DOI] [PubMed] [Google Scholar]
  15. Raina R., Reddy M. A., Ghosal D., Das H. K. Characterization of the gene for the Fe-protein of the vanadium dependent alternative nitrogenase of Azotobacter vinelandii and construction of a Tn5 mutant. Mol Gen Genet. 1988 Sep;214(1):121–127. doi: 10.1007/BF00340189. [DOI] [PubMed] [Google Scholar]
  16. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  17. Robson R. L., Chesshyre J. A., Wheeler C., Jones R., Woodley P. R., Postgate J. R. Genome size and complexity in Azotobacter chroococcum. J Gen Microbiol. 1984 Jul;130(7):1603–1612. doi: 10.1099/00221287-130-7-1603. [DOI] [PubMed] [Google Scholar]
  18. Robson R. L., Postgate J. R. Oxygen and hydrogen in biological nitrogen fixation. Annu Rev Microbiol. 1980;34:183–207. doi: 10.1146/annurev.mi.34.100180.001151. [DOI] [PubMed] [Google Scholar]
  19. Robson R. L., Woodley P. R., Pau R. N., Eady R. R. Structural genes for the vanadium nitrogenase from Azotobacter chroococcum. EMBO J. 1989 Apr;8(4):1217–1224. doi: 10.1002/j.1460-2075.1989.tb03495.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Robson R., Woodley P., Jones R. Second gene (nifH*) coding for a nitrogenase iron protein in Azotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein. EMBO J. 1986 Jun;5(6):1159–1163. doi: 10.1002/j.1460-2075.1986.tb04341.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Ruvkun G. B., Ausubel F. M. Interspecies homology of nitrogenase genes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):191–195. doi: 10.1073/pnas.77.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Strandberg G. W., Wilson P. W. Formation of the nitrogen-fixing enzyme system in Azotobacter vinelandii. Can J Microbiol. 1968 Jan;14(1):25–31. doi: 10.1139/m68-005. [DOI] [PubMed] [Google Scholar]
  23. Thuring R. W., Sanders J. P., Borst P. A freeze-squeeze method for recovering long DNA from agarose gels. Anal Biochem. 1975 May 26;66(1):213–220. doi: 10.1016/0003-2697(75)90739-3. [DOI] [PubMed] [Google Scholar]
  24. Wang S. Z., Chen J. S., Johnson J. L. The presence of five nifH-like sequences in Clostridium pasteurianum: sequence divergence and transcription properties. Nucleic Acids Res. 1988 Jan 25;16(2):439–454. doi: 10.1093/nar/16.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Youvan D. C., Bylina E. J., Alberti M., Begusch H., Hearst J. E. Nucleotide and deduced polypeptide sequences of the photosynthetic reaction-center, B870 antenna, and flanking polypeptides from R. capsulata. Cell. 1984 Jul;37(3):949–957. doi: 10.1016/0092-8674(84)90429-x. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES