Skip to main content

Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW

Molecular and General Genetics MGG volume 225, pages 514–520 (1991)Cite this article

Summary

The nucleotide sequence of a 4.1 kb DNA fragment containing the fixABC region of Azorhizobium caulinodans was established. The three gene products were very similar to the corresponding polypeptides of Rhizobium meliloti. The C-terminal domains of both fixB products displayed a high degree of similarity with the α-subunits of rat and human electron transfer flavoproteins, suggesting a role for the FixB protein in a redox reaction. Two open reading frames (ORF) were found downstream of fixC. The first ORF was identified as fixX on the basis of sequence homology with fixX from several Rhizobium and Bradyrhizobium strains. The second ORF potentially encoded a 69 amino acid product and was found to be homologous to a DNA region in the Rhodobacter capsulatus nif cluster I. Insertion mutagenesis of the A. caulinodans fixX gene conferred a Nif− phenotype to bacteria grown in the free-living state and a Fix− phenotype in symbiotic association with the host plant Sesbania rostrata. A crude extract from the fixX mutant had no nitrogenase activity. Furthermore, data presented in this paper also indicate that the previously identified nifO gene located upstream of fixA was probably a homologue of the nifW gene of Klebsiella pneumoniae and Azotobacter vinelandii.

This is a preview of subscription content, access via your institution.

Access options

Buy single article

Instant access to the full article PDF.

43,34 €

Price includes VAT (Finland)
Tax calculation will be finalised during checkout.

References

  • Arnold W, Rump A, Klipp W, Priefer UB, Pühler A (1988) Nucleotide sequence of a 24206-base-pair DNA fragment carrying the entire nitrogen fixation gene cluster of Klebsiella pneumoniae. J Mol Biol 203:715–738

    Google Scholar 

  • Better M, Lewis B, Corbin D, Ditta G, Helinski DR (1983) Structural relationships among Rhizobium meliloti symbiotic promoters. Cell 35:479–485

    Google Scholar 

  • Beynon J, Cannon M, Buchanan-Wollaston V, Ally A, Setterquist R, Dean D, Cannon F (1988) The nucleotide sequence of the nifT, nifY, nifX and nifW genes of K. pneumoniae. Nucleic Acids Res 16:9860

    Google Scholar 

  • Brushi M, Guerlesquin F (1988) Structure, function and evolution of bacterial ferredoxins. FEMS Microbiol Rev 54:155–176

    Google Scholar 

  • Crane FI, Beinert H (1956) On the mechanism of dehydrogenation of fatty acyl derivatives of coenzyme A. J Biol Chem 218:717–731

    Google Scholar 

  • Denèfle P, Kush A, Norel F, Paquelin A, Elmerich C (1987) Biochemical and genetic analysis of the nifHDKE region of Rhizobium ORS571. Mol Gen Genet 207:280–287

    Google Scholar 

  • Dixon R (1984) The genetic complexity of nitrogen fixation. J Gen Microbiol 130:2745–2755

    Google Scholar 

  • Dreyfus B, Garcia JL, Gillis M (1988) Characterization of Azorhizobium caulinodans gen nov, a stem nodulating nitrogen fixing bacterium isolated from Sesbania rostrata. Int J Syst Bacteriol 38:89–98

    Google Scholar 

  • Dusha I, Kovalenko S, Banfalvi Z, Kondorosi A (1987) Rhizobium meliloti insertion element ISRm2 and its use for identification of the fixX gene. J Bacteriol 169:1403–1409

    Google Scholar 

  • Earl CD, Ronson CW, Ausubel FM (1987) Genetic and structural analysis of the Rhizobium meliloti fixA, fixB, fixC, and fixX genes. J Bacteriol 169:1127–1136

    Google Scholar 

  • Elmerich C, Dreyfus BL, Reysset G, Aubert JP (1982) Genetic analysis of nitrogen fixation in a tropical fast-growing Rhizobium. EMBO J 1:499–503

    Google Scholar 

  • Evans D, Jones R, Woodley P, Robson R (1988) Further analysis of nitrogen fixation (nif) genes in Azotobacter chroococcum: Identification and expression in Klebsiella pneumoniae of nifS, ni V, nifM, and nifB genes and localization of nifE/N-, nifU-, nifA- and fixABC-like genes. J Gen Microbiol 134:931–942

    Google Scholar 

  • Finocchiaro G, Michinori I, Yasuyuki I, Tanaka K (1988) Molecular cloning and nucleotide sequence of cDNAs encoding the α-subunit of human electron transfer protein. J Biol Chem 263:15773–15780

    Google Scholar 

  • Fogher C, Dusha I, Barbot P, Elmerich C (1985) Heterologous hybridization of Azospirillum DNA to Rhizobium nod and fix genes. FEMS Microbiol Lett 30:245–249

    Google Scholar 

  • Fuhrmann M, Fischer HM, Hennecke H (1985) Mapping of Rhizobium japonicum nifB-, fixBC-, and fixA-like genes and identification of the fixA promoter. Mol Gen Genet 199:315–322

    Google Scholar 

  • Galimand M, Perroud B, Delorme F, Paquelin A, Vieille C, Bozouklian H, Elmerich C (1989) Identification of DNA regions homologous to nitrogen fixation genes nifE, nifuS and fixABC in Azospirillum brasilense Sp7. J Gen Microbiol 135:1047–1059

    Google Scholar 

  • Grönger P, Manian S, Reiländer H, O'Connel M, Priefer UB, Pühler A (1987) Organization and partial sequence of a DNA region of the Rhizobium leguminosarum symbiotic plasmid containing the fixABC, nifA, nifB and a novel open reading frame. Nucleic Acids Res 15:31–49

    Google Scholar 

  • Gubler M, Hennecke H (1986) fixA, B and C genes are essential for symbiotic and free-living, microaerobic nitrogen fixation. FEBS Lett 200:186–192

    Google Scholar 

  • Gubler M, Hennecke H (1988) Regulation of the fixA gene and fixBC operon in Bradyrhizobium japonicum. J Bacteriol 170:1205–1214

    Google Scholar 

  • Gubler M, Zürcher T, Hennecke H (1989) The Bradhyrhizobium japonicum fixBCX operon: Identification of fixX and of a 5′ mRNA region affecting the level of the fixBCX transcript. Mol Microbiol 3:141–148

    Google Scholar 

  • Haselkorn R, Buikema WJ (1991) Nitrogen fixation in Cyanobacteria. In: Stacey G, Evans HJ, Burris RH (eds) Biological nitrogen fixation. Routledge, Chapman and Hall, New York London, in press

    Google Scholar 

  • Iismaa SE, Watson JM (1987) A gene upstream of the Rhizobium trifolii nifA gene encodes a ferredoxin-like protein. Nucleic Acids Res 15:3180

    Google Scholar 

  • Iismaa SE, Ealing PM, Scott KF, Watson JM (1989) Molecular linkage of the nif/fix and nod gene regions in Rhizobium leguminosarum biovar trifolii. Mol Microbiol 3:1753–1764

    Google Scholar 

  • Jacobson MR, Brigle KV, Bennett LT, Setterquist RA, Wilson MS, Cash VL, Beynon J, Newton WE, Dean DR (1989a) Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii. J Bacteriol 171:1017–1027

    Google Scholar 

  • Jacobson MR, Cash VL, Weiss MC, Laird NF, Newton WE, Dean DR (1989b) Biochemical and genetic analysis of the nifUSVWZM cluster from Azotobacter vinelandii. Mol Gen Genet 219:49–57

    CAS  PubMed  Google Scholar 

  • Kaminski PA, Norel F, Desnoues N, Kush A, Salzano G, Elmerich C (1988) Characterization of the fixABC region of Azorhizobium caulinodans ORS571 and identification of a new nitrogen fixation gene. Mol Gen Genet 214:496–502

    Google Scholar 

  • Moreno-Vivian C, Hennecke S, Pühler A, Klipp W (1989) Open reading frame 5 (ORF5), encoding a ferredoxin-like protein, and nifQ are cotranscribed with nifE, nifty, nifX and ORF4 in Rhodobacter capsulatus. J Bacteriol 171:2591–2598

    Google Scholar 

  • Norel F, Elmerich C (1987) Nucleotide sequence and functional analysis of the two nifH copies of Rhizobium ORS571. J Gen Microbiol 133:1563–1576

    Google Scholar 

  • Paul W, Merrick M (1989) The roles of the nifW, nifZ and nifM genes of Klebsiella pneumoniae in nitrogenase biosynthesis. Eur J Biochem 178:675–682

    Google Scholar 

  • Pühler A, Aguilar MO, Hynes M, Müller P, Klipp W, Priefer U, Simon R, Weber G (1984) Advances in the genetics of freeliving and symbiotic nitrogen fixing bacteria. In: Veeger C, Newton WE (eds) Advances in nitrogen fixation research. Martinus Nijhoff DRW Junk Publishers, The Hague Boston Lancaster Pudoc Wageningen, p 609

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain termination inhibitors. Proc Natl Acad Sci USA 74:5463–5467

    CAS  PubMed  Google Scholar 

  • Shine J, Dalgarno L (1974) The 3′-terminal sequence of Escherichia coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346

    Google Scholar 

  • Shinzawa K, Inagaki T, Ohishi N, Ichihara C, Tsukagoshi N, Udaka S, Yagi K (1988) Molecular cloning of a cDNA for α-subunit of rat liver electron transfer protein. Biochem Biophys Res Commun 155:300–304

    Google Scholar 

  • Staden R (1983) Computer methods for DNA sequencers. In: Hindley J, Worth TS, Bividon RH (eds) DNA sequencing. Elsevier, Amsterdam, pp 311–368

    Google Scholar 

Download references

Author information

Affiliations

  1. Unité de Physiologic Cellulaire and CNRS URA 1300, Département des Biotechnologies, Institut Pasteur, 25 rue du Docteur Roux, F-75724, Paris Cedex 15, France

    Fabrizio Arigoni, P. Alexandre Kaminski & Claudine Elmerich

  2. Eidgenössische Technische Hochschule, ETH-Zentrum, Mikrobiologisches Institut, Schmelzbergstrasse 7, CH-8092, Zürich, Switzerland

    Hauke Hennecke

Authors
  1. Fabrizio Arigoni
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. P. Alexandre Kaminski
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Hauke Hennecke
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Claudine Elmerich
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

Communicated by A. Kondorosi

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Arigoni, F., Kaminski, P.A., Hennecke, H. et al. Nucleotide sequence of the fixABC region of Azorhizobium caulinodans ORS571: similarity of the fixB product with eukaryotic flavoproteins, characterization of fixX, and identification of nifW . Molec. Gen. Genet. 225, 514–520 (1991). https://doi.org/10.1007/BF00261695

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00261695

Key words

  • Azorhizobium caulinodans
  • Electron transfer flavoprotein
  • nifOfixABCX genes
  • nifW homologue
  • Nitrogen fixation