Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1983 May;80(10):3005–3009. doi: 10.1073/pnas.80.10.3005

Organization and expression of Rhizobium meliloti nitrogen fixation genes

David Corbin 1, Leslie Barran 1,*, Gary Ditta 1
PMCID: PMC393962  PMID: 16593313

Abstract

The boundaries of a nif gene cluster in Rhizobium meliloti were determined by Tn5 mutagenesis. These genes are clustered within a 14- to 15-kilobase (kb) region that includes the nitrogenase structural genes. Sequences within 10 kb on either side of this region are devoid of symbiotically essential gene function. RNA blot analysis identified a 5- to 6-kb transcript that corresponds to the nitrogenase structural gene operon. The 5′ end of this transcript and its polarity were determined by nuclease S1 mapping. The 5′ end of another nif transcript was also identified by nuclease S1 mapping. The promoter regions for these two nif transcripts control transcription in divergent directions and are separated by 1.9 kb of symbiotically unessential DNA. One Tn5 insertion within the nitrogenase operon did not create a polar mutation. The implications of this finding and the overall emerging picture of the genetic organization of this nif region are discussed.

Keywords: nif gene, Tn5 mutagenesis, nuclease S1 mapping

Full text

PDF
3005

Images in this article

3007Image
on p.3007
3007Image
on p.3007
3007Image
on p.3007
3007Image
on p.3007

Selected References

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

  1. Berg D. E., Weiss A., Crossland L. Polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol. 1980 May;142(2):439–446. doi: 10.1128/jb.142.2.439-446.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  3. Bánfalvi Z., Sakanyan V., Koncz C., Kiss A., Dusha I., Kondorosi A. Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet. 1981;184(2):318–325. doi: 10.1007/BF00272925. [DOI] [PubMed] [Google Scholar]
  4. Cannon F. C., Riedel G. E., Ausubel F. M. Overlapping sequences of Klebsiella pneumoniae nifDNA cloned and characterized. Mol Gen Genet. 1979 Jul 2;174(1):59–66. doi: 10.1007/BF00433306. [DOI] [PubMed] [Google Scholar]
  5. Casey J., Davidson N. Rates of formation and thermal stabilities of RNA:DNA and DNA:DNA duplexes at high concentrations of formamide. Nucleic Acids Res. 1977;4(5):1539–1552. doi: 10.1093/nar/4.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Corbin D., Ditta G., Helinski D. R. Clustering of nitrogen fixation (nif) genes in Rhizobium meliloti. J Bacteriol. 1982 Jan;149(1):221–228. doi: 10.1128/jb.149.1.221-228.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Israel D. W., Howard R. L., Evans H. J., Russell S. A. Purification and characterization of the molybdenum-iron protein component of nitrogenase from soybean nodule bacteroids. J Biol Chem. 1974 Jan 25;249(2):500–508. [PubMed] [Google Scholar]
  9. Kassavetis G. A., Geiduschek E. P. Bacteriophage T4 late promoters: mapping 5' ends of T4 gene 23 mRNAs. EMBO J. 1982;1(1):107–114. doi: 10.1002/j.1460-2075.1982.tb01132.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Leong S. A., Ditta G. S., Helinski D. R. Heme biosynthesis in Rhizobium. Identification of a cloned gene coding for delta-aminolevulinic acid synthetase from Rhizobium meliloti. J Biol Chem. 1982 Aug 10;257(15):8724–8730. [PubMed] [Google Scholar]
  11. Loening U. E. Molecular weights of ribosomal RNA in relation to evolution. J Mol Biol. 1968 Dec;38(3):355–365. doi: 10.1016/0022-2836(68)90391-4. [DOI] [PubMed] [Google Scholar]
  12. MacNeil D., Zhu J., Brill W. J. Regulation of nitrogen fixation in Klebsiella pneumoniae: isolation and characterization of strains with nif-lac fusions. J Bacteriol. 1981 Jan;145(1):348–357. doi: 10.1128/jb.145.1.348-357.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  14. Meade H. M., Long S. R., Ruvkun G. B., Brown S. E., Ausubel F. M. Physical and genetic characterization of symbiotic and auxotrophic mutants of Rhizobium meliloti induced by transposon Tn5 mutagenesis. J Bacteriol. 1982 Jan;149(1):114–122. doi: 10.1128/jb.149.1.114-122.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Prakash R. K., Schilperoort R. A., Nuti M. P. Large plasmids of fast-growing rhizobia: homology studies and location of structural nitrogen fixation (nif) genes. J Bacteriol. 1981 Mar;145(3):1129–1136. doi: 10.1128/jb.145.3.1129-1136.1981. [DOI] [PMC free article] [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. Roberts G. P., Brill W. J. Genetics and regulation of nitrogen fixation. Annu Rev Microbiol. 1981;35:207–235. doi: 10.1146/annurev.mi.35.100181.001231. [DOI] [PubMed] [Google Scholar]
  18. Rosenberg C., Boistard P., Dénarié J., Casse-Delbart F. Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet. 1981;184(2):326–333. doi: 10.1007/BF00272926. [DOI] [PubMed] [Google Scholar]
  19. Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
  20. Ruvkun G. B., Sundaresan V., Ausubel F. M. Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes. Cell. 1982 Jun;29(2):551–559. doi: 10.1016/0092-8674(82)90171-4. [DOI] [PubMed] [Google Scholar]
  21. Scott D. B., Hennecke H., Lim S. T. The biosynthesis of nitrogenase MoFe protein polypeptides in free-living cultures of Rhizobium japonicum. Biochim Biophys Acta. 1979 Dec 17;565(2):365–378. doi: 10.1016/0005-2787(79)90212-0. [DOI] [PubMed] [Google Scholar]
  22. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  23. Thomas M., White R. L., Davis R. W. Hybridization of RNA to double-stranded DNA: formation of R-loops. Proc Natl Acad Sci U S A. 1976 Jul;73(7):2294–2298. doi: 10.1073/pnas.73.7.2294. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Török I., Kondorosi A. Nucleotide sequence of the R.meliloti nitrogenase reductase (nifH) gene. Nucleic Acids Res. 1981 Nov 11;9(21):5711–5723. doi: 10.1093/nar/9.21.5711. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Whiting M. J., Dilworth M. J. Legume root nodule nitrogenase. Purification, properties, and studies on its genetic control. Biochim Biophys Acta. 1974 Dec 18;371(2):337–351. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES