Skip to main content
PMC home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1993 Jun;59(6):1883–1886. doi: 10.1128/aem.59.6.1883-1886.1993

Presence of a Vanadium Nitrogenase in Azotobacter paspali

Elazar Fallik 1,†, Peter G Hartel 1, Robert L Robson 1,*
PMCID: PMC182175  PMID: 16348965

Abstract

There have been no previous studies on the genetics of Azotobacter paspali, an aerobic bacterium which forms a highly specific diazotrophic association with Bahia grass (Paspalum notatum). We constructed A. paspali strains defective in the molybdenum nitrogenase so that alternative N2ases could be studied. The cosmid vector pTBE and genomic DNA fragments (∼50 kb) of A. paspali ATCC 23367 were used to construct a gene library in Escherichia coli. Recombinant cosmids containing sequences homologous to molybdenum nitrogenase nifDK structural genes were identified by hybridization. A 2.9-kb fragment bearing the putative nifDK genes of A. paspali was subcloned and mutagenized in vitro by the insertion of a kanamycin resistance gene cassette. The mutation was recombined into the chromosome of A. paspali with the suicide vector pCU101. One resultant mutant strain, AP2, was incapable of diazotrophic growth in a molybdenum-containing medium (Nif-) without vanadium but grew well in a molybdenum-deficient medium with vanadium. The nitrogenase system in AP2 reduced acetylene to ethylene and produced ethane as 2.4% of the total products. Molybdenum levels as low as 10 nM prevented the diazotrophic growth of AP2, even in the presence of vanadium at levels up to 10 μM. These results are consistent with the existence of a vanadium nitrogenase system in A. paspali.

Full text

PDF
1886

Selected References

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

  1. 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]
  2. 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]
  3. Dagert M., Ehrlich S. D. Prolonged incubation in calcium chloride improves the competence of Escherichia coli cells. Gene. 1979 May;6(1):23–28. doi: 10.1016/0378-1119(79)90082-9. [DOI] [PubMed] [Google Scholar]
  4. Fallik E., Chan Y. K., Robson R. L. Detection of alternative nitrogenases in aerobic gram-negative nitrogen-fixing bacteria. J Bacteriol. 1991 Jan;173(1):365–371. doi: 10.1128/jb.173.1.365-371.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Grosveld F. G., Lund T., Murray E. J., Mellor A. L., Dahl H. H., Flavell R. A. The construction of cosmid libraries which can be used to transform eukaryotic cells. Nucleic Acids Res. 1982 Nov 11;10(21):6715–6732. doi: 10.1093/nar/10.21.6715. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. Jacobson M. R., Premakumar R., Bishop P. E. Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii. J Bacteriol. 1986 Aug;167(2):480–486. doi: 10.1128/jb.167.2.480-486.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Joerger R. D., Bishop P. E. Bacterial alternative nitrogen fixation systems. Crit Rev Microbiol. 1988;16(1):1–14. doi: 10.3109/10408418809104465. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Lacks S., Greenberg B. Complementary specificity of restriction endonucleases of Diplococcus pneumoniae with respect to DNA methylation. J Mol Biol. 1977 Jul;114(1):153–168. doi: 10.1016/0022-2836(77)90289-3. [DOI] [PubMed] [Google Scholar]
  11. Mead D. A., Szczesna-Skorupa E., Kemper B. Single-stranded DNA 'blue' T7 promoter plasmids: a versatile tandem promoter system for cloning and protein engineering. Protein Eng. 1986 Oct-Nov;1(1):67–74. doi: 10.1093/protein/1.1.67. [DOI] [PubMed] [Google Scholar]
  12. Messing J., Gronenborn B., Müller-Hill B., Hans Hopschneider P. Filamentous coliphage M13 as a cloning vehicle: insertion of a HindII fragment of the lac regulatory region in M13 replicative form in vitro. Proc Natl Acad Sci U S A. 1977 Sep;74(9):3642–3646. doi: 10.1073/pnas.74.9.3642. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Murray N. E., Brammar W. J., Murray K. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol Gen Genet. 1977 Jan 7;150(1):53–61. doi: 10.1007/BF02425325. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. 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]
  16. 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]
  17. Schneider K., Müller A., Johannes K. U., Diemann E., Kottmann J. Selective removal of molybdenum traces from growth media of N2-fixing bacteria. Anal Biochem. 1991 Mar 2;193(2):292–298. doi: 10.1016/0003-2697(91)90024-n. [DOI] [PubMed] [Google Scholar]
  18. Thatte V., Iyer V. N. Cloning of a plasmid region specifying the N transfer system of bacterial conjugation in Escherichia coli. Gene. 1983 Mar;21(3):227–236. doi: 10.1016/0378-1119(83)90006-9. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES