Skip to main content
PMC 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
. 1984 Dec;81(23):7358–7362. doi: 10.1073/pnas.81.23.7358

Structural genes of dinitrogenase and dinitrogenase reductase are transcribed from two separate promoters in the broad host range cowpea Rhizobium strain IRc78

Allen C Yun 1, Aladar A Szalay 1,*
PMCID: PMC392145  PMID: 16578778

Abstract

The nucleotide sequence of the structural gene (nifD) coding for the α-subunit of dinitrogenase along with its flanking sequences has been determined in cowpea Rhizobium IRc78. The coding sequence consists of 1500 nucleotides, which corresponds to a predicted amino acid sequence of 500 residues and a molecular weight of 56,025. Nucleotide homology to nifD from the blue-green alga, Anabaena, and Parasponia Rhizobium, are 63% and 90%, respectively. Cowpea Rhizobium IRc78 nifD and nifK (encodes the β-subunit of dinitrogenase) genes are linked, separated by 69 nucleotides. In contrast to fast-growing rhizobia, the structural genes of dinitrogenase (nifDK) are transcribed from a different promoter than the structural gene of dinitrogenase reductase (nifH). Transcription of nifDK initiates 41 nucleotides upstream of the start codon for the nifDK operon. Two transcription initiation sites, localized at 152 and 114 nucleotides upstream of the start codon, were determined for the nifH operon. Two nucleotide sequences, a hexamer (G-G-T-T-G-C) and a pentamer (T-G-G-C-A), centered at approximately -15 and -25, respectively, are conserved in the nifD and nifH promoter regions and are not present in the 69-nucleotide nifDK junction. No sequence homology other than a possible ribosome binding site, T-T-G-A-[unk]-G-G-A, located 14 nucleotides upstream of the initiation codon was detected between the transcribed but untranslated leader regions of nifD and nifH.

Keywords: S1 nuclease mapping, nucleotide sequence, nitrogen fixation, protein sequence conservation

Full text

PDF

Images in this article

7361Image
on p.7361
7361Image
on p.7361

Selected References

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

  1. Adams T. H., Chelm B. K. The nifH and nifDK promoter regions from Rhizobium japonicum share structural homologies with each other and with nitrogen-regulated promoters from other organisms. J Mol Appl Genet. 1984;2(4):392–405. [PubMed] [Google Scholar]
  2. Ausubel F. M. Regulation of nitrogen fixation genes. Cell. 1984 May;37(1):5–6. doi: 10.1016/0092-8674(84)90294-0. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. Bernard H. U., Helinski D. R. Use of the lambda phage promoter PL to promote gene expression in hybrid plasmid cloning vehicles. Methods Enzymol. 1979;68:482–492. doi: 10.1016/0076-6879(79)68037-0. [DOI] [PubMed] [Google Scholar]
  5. Better M., Lewis B., Corbin D., Ditta G., Helinski D. R. Structural relationships among Rhizobium meliloti symbiotic promoters. Cell. 1983 Dec;35(2 Pt 1):479–485. doi: 10.1016/0092-8674(83)90181-2. [DOI] [PubMed] [Google Scholar]
  6. Beynon J., Cannon M., Buchanan-Wollaston V., Cannon F. The nif promoters of Klebsiella pneumoniae have a characteristic primary structure. Cell. 1983 Sep;34(2):665–671. doi: 10.1016/0092-8674(83)90399-9. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Hadley R. G., Eaglesham A. R., Szalay A. A. Conservation of DNA regions adjacent to nifKDH homologous sequences in diverse slow-growing Rhizobium strains. J Mol Appl Genet. 1983;2(3):225–236. [PubMed] [Google Scholar]
  10. Hase T., Nakano T., Matsubara H., Zumft W. G. Correspondence of the larger subunit of the MoFe-protein in clostridial nitrogenase to the nif D gene products of other N2-fixing organisms. J Biochem. 1981 Jul;90(1):295–298. doi: 10.1093/oxfordjournals.jbchem.a133466. [DOI] [PubMed] [Google Scholar]
  11. Hausinger R. P., Howard J. B. Comparison of the iron proteins from the nitrogen fixation complexes of Azotobacter vinelandii, Clostridium pasteurianum, and Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3826–3830. doi: 10.1073/pnas.77.7.3826. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kaluza K., Fuhrmann M., Hahn M., Regensburger B., Hennecke H. In Rhizobium japonicum the nitrogenase genes nifH and nifDK are separated. J Bacteriol. 1983 Aug;155(2):915–918. doi: 10.1128/jb.155.2.915-918.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Lammers P. J., Haselkorn R. Sequence of the nifD gene coding for the alpha subunit of dinitrogenase from the cyanobacterium Anabaena. Proc Natl Acad Sci U S A. 1983 Aug;80(15):4723–4727. doi: 10.1073/pnas.80.15.4723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lundell D. J., Howard J. B. Isolation and sequences of the cysteinyl tryptic peptides from the MoFe-protein of Azotobacter vinelandii nitrogenase. J Biol Chem. 1981 Jun 25;256(12):6385–6391. [PubMed] [Google Scholar]
  15. Masterson R. V., Russell P. R., Atherly A. G. Nitrogen fixation (nif) genes and large plasmids of Rhizobium japonicum. J Bacteriol. 1982 Nov;152(2):928–931. doi: 10.1128/jb.152.2.928-931.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  17. Mazur B. J., Chui C. F. Sequence of the gene coding for the beta-subunit of dinitrogenase from the blue-green alga Anabaena. Proc Natl Acad Sci U S A. 1982 Nov;79(22):6782–6786. doi: 10.1073/pnas.79.22.6782. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Mazur B. J., Rice D., Haselkorn R. Identification of blue-green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):186–190. doi: 10.1073/pnas.77.1.186. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Merrick M., Filser M., Dixon R., Elmerich C., Sibold L., Houmard J. The use of translocatable genetic elements to construct a fine-structure map of the Klebsiella pneumoniae nitrogen fixation (nif) gene cluster. J Gen Microbiol. 1980 Apr;117(2):509–520. doi: 10.1099/00221287-117-2-509. [DOI] [PubMed] [Google Scholar]
  20. Mevarech M., Rice D., Haselkorn R. Nucleotide sequence of a cyanobacterial nifH gene coding for nitrogenase reductase. Proc Natl Acad Sci U S A. 1980 Nov;77(11):6476–6480. doi: 10.1073/pnas.77.11.6476. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mortenson L. E., Thorneley R. N. Structure and function of nitrogenase. Annu Rev Biochem. 1979;48:387–418. doi: 10.1146/annurev.bi.48.070179.002131. [DOI] [PubMed] [Google Scholar]
  22. Ow D. W., Sundaresan V., Rothstein D. M., Brown S. E., Ausubel F. M. Promoters regulated by the glnG (ntrC) and nifA gene products share a heptameric consensus sequence in the -15 region. Proc Natl Acad Sci U S A. 1983 May;80(9):2524–2528. doi: 10.1073/pnas.80.9.2524. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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]
  24. Rice D., Mazur B. J., Haselkorn R. Isolation and physical mapping of nitrogen fixation genes from the cyanobacterium Anabaena 7120. J Biol Chem. 1982 Nov 10;257(21):13157–13163. [PubMed] [Google Scholar]
  25. Riedel G. E., Ausubel F. M., Cannon F. C. Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2866–2870. doi: 10.1073/pnas.76.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. 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]
  28. Schetgens T. M., Bakkeren G., van Dun C., Hontelez J. G., van den Bos R. C., van Kammen A. Molecular cloning and functional characterization of Rhizobium leguminosarum structural nif-genes by site-directed transposon mutagenesis and expression in Escherichia coli minicells. J Mol Appl Genet. 1984;2(4):406–421. [PubMed] [Google Scholar]
  29. Scott K. F., Rolfe B. G., Shine J. Biological nitrogen fixation: primary structure of the Klebsiella pneumoniae nifH and nifD genes. J Mol Appl Genet. 1981;1(1):71–81. [PubMed] [Google Scholar]
  30. Scott K. F., Rolfe B. G., Shine J. Biological nitrogen fixation: primary structure of the Rhizobium trifolii iron protein gene. DNA. 1983;2(2):149–155. doi: 10.1089/dna.1983.2.149. [DOI] [PubMed] [Google Scholar]
  31. Scott K. F., Rolfe B. G., Shine J. Nitrogenase structural genes are unlinked in the nonlegume symbiont Parasponia rhizobium. DNA. 1983;2(2):141–148. doi: 10.1089/dna.1983.2.141. [DOI] [PubMed] [Google Scholar]
  32. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Sundaresan V., Ausubel F. M. Nucleotide sequence of the gene coding for the nitrogenase iron protein from Klebsiella pneumoniae. J Biol Chem. 1981 Mar 25;256(6):2808–2812. [PubMed] [Google Scholar]
  34. 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]
  35. Tanaka M., Haniu M., Yasunobu K. T. The amino acid sequence of Clostridium pasteurianum iron protein, a component of nitrogenase. III. The NH2-terminal and COOH-terminal sequences, tryptic peptides of large cyanogen bromide peptides, and the complete sequence. J Biol Chem. 1977 Oct 25;252(20):7093–7100. [PubMed] [Google Scholar]
  36. 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]
  37. Zimmerman J. L., Szeto W. W., Ausubel F. M. Molecular characterization of Tn5-induced symbiotic (Fix-) mutants of Rhizobium meliloti. J Bacteriol. 1983 Dec;156(3):1025–1034. doi: 10.1128/jb.156.3.1025-1034.1983. [DOI] [PMC free article] [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