Skip to main content
PMC home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1986 Jan;165(1):72–81. doi: 10.1128/jb.165.1.72-81.1986

Characterization of three genomic loci encoding Rhizobium sp. strain ORS571 N2 fixation genes.

R G Donald, D W Nees, C K Raymond, A I Loroch, R A Ludwig
PMCID: PMC214372  PMID: 3001035

Abstract

Sixty-five independent, N2 fixation-defective (Nif-) vector insertion (Vi) mutants were selected, cloned, and mapped to the ORS571 genome. The recombinant Nif::Vi plasmids obtained in this way were used as DNA hybridization probes to isolate homologous phages from a genomic library of ORS571 constructed in lambda EMBL3. Genomic maps were drawn for three ORS571 Nif gene loci. Forty-five Nif::Vi mutants in genomic Nif locus 1 defined two gene clusters separated by 8 kilobase pairs (kb) of DNA. In the first cluster, 36 Nif::Vi mutants mapped to a 7-kb DNA segment that showed DNA homology with Klebsiella pneumoniae nifHDKE and encoded at least two Nif operons. In the other cluster, nine Nif::Vi mutants mapped to a 1.5-kb DNA segment that showed homology with K. pneumoniae and Rhizobium meliloti nifA; this DNA segment encoded a separate Nif operon. Fifteen Nif::Vi mutants mapped to a 3.5-kb DNA segment defined as Nif locus 2 and showed DNA homology with the R. meliloti P2 fixABC operon. Nif locus 2 carries a second nifH (nifH2) gene. Four Nif::Vi mutants mapped to a 2-kb DNA segment defined as Nif locus 3 and showed DNA homology with K. pneumoniae nifB. DNA from lambda Nif phages comprising all three genomic Nif loci was subcloned in plasmid vectors able to stably replicate in ORS571. These plasmid subclones were introduced into ORS571 strains carrying physically mapped Nif::Vi insertions, and genetic complementations were conducted. With the exception of certain mutants mapping to the nifDK genes, all mutants could be complemented to Nif+ when they carried plasmid subclones of defined genomic DNA regions. Conversely, most nifDK mutants behaved as pseudodominant alleles.

Full text

PDF

Selected References

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

  1. Adams T. H., McClung C. R., Chelm B. K. Physical organization of the Bradyrhizobium japonicum nitrogenase gene region. J Bacteriol. 1984 Sep;159(3):857–862. doi: 10.1128/jb.159.3.857-862.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Buikema W. J., Szeto W. W., Lemley P. V., Orme-Johnson W. H., Ausubel F. M. Nitrogen fixation specific regulatory genes of Klebsiella pneumoniae and Rhizobium meliloti share homology with the general nitrogen regulatory gene ntrC of K. pneumoniae. Nucleic Acids Res. 1985 Jun 25;13(12):4539–4555. doi: 10.1093/nar/13.12.4539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. 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]
  4. Corbin D., Barran L., Ditta G. Organization and expression of Rhizobium meliloti nitrogen fixation genes. Proc Natl Acad Sci U S A. 1983 May;80(10):3005–3009. doi: 10.1073/pnas.80.10.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Ditta G., Schmidhauser T., Yakobson E., Lu P., Liang X. W., Finlay D. R., Guiney D., Helinski D. R. Plasmids related to the broad host range vector, pRK290, useful for gene cloning and for monitoring gene expression. Plasmid. 1985 Mar;13(2):149–153. doi: 10.1016/0147-619x(85)90068-x. [DOI] [PubMed] [Google Scholar]
  6. 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]
  7. Donald R. G., Ludwig R. A. Rhizobium sp. strain ORS571 ammonium assimilation and nitrogen fixation. J Bacteriol. 1984 Jun;158(3):1144–1151. doi: 10.1128/jb.158.3.1144-1151.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Donald R. G., Raymond C. K., Ludwig R. A. Vector insertion mutagenesis of Rhizobium sp. strain ORS571: direct cloning of mutagenized DNA sequences. J Bacteriol. 1985 Apr;162(1):317–323. doi: 10.1128/jb.162.1.317-323.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Downie J. A., Ma Q. S., Knight C. D., Hombrecher G., Johnston A. W. Cloning of the symbiotic region of Rhizobium leguminosarum: the nodulation genes are between the nitrogenase genes and a nifA-like gene. EMBO J. 1983;2(6):947–952. doi: 10.1002/j.1460-2075.1983.tb01526.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elmerich C., Dreyfus B. L., Reysset G., Aubert J. P. Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium. EMBO J. 1982;1(4):499–503. doi: 10.1002/j.1460-2075.1982.tb01197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  12. Frischauf A. M., Lehrach H., Poustka A., Murray N. Lambda replacement vectors carrying polylinker sequences. J Mol Biol. 1983 Nov 15;170(4):827–842. doi: 10.1016/s0022-2836(83)80190-9. [DOI] [PubMed] [Google Scholar]
  13. Golden J. W., Robinson S. J., Haselkorn R. Rearrangement of nitrogen fixation genes during heterocyst differentiation in the cyanobacterium Anabaena. Nature. 1985 Apr 4;314(6010):419–423. doi: 10.1038/314419a0. [DOI] [PubMed] [Google Scholar]
  14. 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]
  15. Merrick M. J. A new model for nitrogen control. Nature. 1982 Jun 3;297(5865):362–363. doi: 10.1038/297362a0. [DOI] [PubMed] [Google Scholar]
  16. Quinto C., De La Vega H., Flores M., Leemans J., Cevallos M. A., Pardo M. A., Azpiroz R., De Lourdes Girard M., Calva E., Palacios R. Nitrogenase reductase: A functional multigene family in Rhizobium phaseoli. Proc Natl Acad Sci U S A. 1985 Feb;82(4):1170–1174. doi: 10.1073/pnas.82.4.1170. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Reibach P. H., Mask P. L., Streeter J. G. A rapid one-step method for the isolation of bacteroids from root nodules of soybean plants, utilizing self-generating Percoll gradients. Can J Microbiol. 1981 May;27(5):491–495. doi: 10.1139/m81-072. [DOI] [PubMed] [Google Scholar]
  18. Roberts G. P., MacNeil T., MacNeil D., Brill W. J. Regulation and characterization of protein products coded by the nif (nitrogen fixation) genes of Klebsiella pneumoniae. J Bacteriol. 1978 Oct;136(1):267–279. doi: 10.1128/jb.136.1.267-279.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. 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]
  20. Scolnik P. A., Haselkorn R. Activation of extra copies of genes coding for nitrogenase in Rhodopseudomonas capsulata. Nature. 1984 Jan 19;307(5948):289–292. doi: 10.1038/307289a0. [DOI] [PubMed] [Google Scholar]
  21. Singh L., Jones K. W. The use of heparin as a simple cost-effective means of controlling background in nucleic acid hybridization procedures. Nucleic Acids Res. 1984 Jul 25;12(14):5627–5638. doi: 10.1093/nar/12.14.5627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Szeto W. W., Zimmerman J. L., Sundaresan V., Ausubel F. M. A Rhizobium meliloti symbiotic regulatory gene. Cell. 1984 Apr;36(4):1035–1043. doi: 10.1016/0092-8674(84)90053-9. [DOI] [PubMed] [Google Scholar]
  23. Tsien H. C., Dreyfus B. L., Schmidt E. L. Initial stages in the morphogenesis of nitrogen-fixing stem nodules of Sesbania rostrata. J Bacteriol. 1983 Nov;156(2):888–897. doi: 10.1128/jb.156.2.888-897.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Yun A. C., Szalay A. A. Structural genes of dinitrogenase and dinitrogenase reductase are transcribed from two separate promoters in the broad host range cowpea Rhizobium strain IRc78. Proc Natl Acad Sci U S A. 1984 Dec;81(23):7358–7362. doi: 10.1073/pnas.81.23.7358. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. de Vries G. E., Raymond C. K., Ludwig R. A. Extension of bacteriophage lambda host range: selection, cloning, and characterization of a constitutive lambda receptor gene. Proc Natl Acad Sci U S A. 1984 Oct;81(19):6080–6084. doi: 10.1073/pnas.81.19.6080. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES