Skip to main content
SpringerLink
Log in

Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN ofRhizobium meliloti involved in symbiotic nitrogen fixation

  • Published:
Molecular and General Genetics MGG Aims and scope Submit manuscript

Summary

TheRhizobium meliloti fdxN gene, which is part of thenifA-nifB fdxN operon, is absolutely required for symbiotic nitrogen fixation. The deduced sequence of the FdxN protein is characterized by two cysteine motifs typical of bacterial-type ferredoxins. The Fix− phenotype of anR. meliloti fdxN: :[Tc] mutant could be rescued by theR. leguminosarum fdxN gene, whereas no complementation was observed withnif-associated genes encoding ferredoxins fromBradyrhizobium japonicum, Azotobacter vinelandii, A. chroococcum andRhodobacter capsulatus. In addition to these heterologous genes, severalR. meliloti fdxN mutant genes constructed by site-directed mutagenesis were analyzed. Not only a cysteine residue within the second cysteine motif (position 42), which is known to coordinate the Fe-S cluster in homologous proteins, but also a cysteine located down-stream of this motif (position 61), was found to be essential for the activity of theR. meliloti FdxN protein. Changing the amino acid residue proline in position 56 into methionine resulted in a FdxN mutant protein with decreased activity, whereas changes in positions 35 (Asp35Glu) and 45 (Gly45Glu) had no significant effect on the function of the FdxN mutant proteins. In contrast to bacterial-type ferredoxins, which contain two identical cysteine motifs of the form C-X2-C-X2-C-X3-C,nif-associated ferredoxins, includingR. meliloti FdxN, are characterized by two different cysteine motifs. Six “additional” amino acids separate the second (Cys42) and the third cysteine (Cys51) in the C-terminal motif (C-X2-C-X8-C-X3-C). By molecular modelling, it was predicted that these amino acid residues form a loop, which does not alter the relative positions of the neighbouring cysteines. Deletion of this loop resulted in anR. meliloti FdxN mutant protein, which exhibited almost 70% wild-type activity, indicating that the predicted loop does not affect Fe-S cluster binding and plays no crucial role in activity of the FdxN protein.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Adman ET, Sieker LC, Jensen LH (1973) The structure of a bacterial ferredoxin. J Biol Chem 248:3987–3996

    Google Scholar 

  • Aguilar OM, Kapp D, Pühler A (1985) Characterisation of aRhizobium meliloti fixation gene (fixF) located near the common nodulation region. J Bacteriol 164:245–254

    Google Scholar 

  • Aguilar OM, Reiländer H, Arnold W, Pühler A (1987)Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by anifA-dependent promoter and codes for a polypeptide homologous to thenifK gene product. J Bacteriol 169:5393–5400

    Google Scholar 

  • Beaucage SL, Caruthers MH (1981) Deoxynucleoside phosphoramidites — a new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett 22:1859–1862

    Google Scholar 

  • Buikema WJ, Szeto WW, Lemley PV, Orme-Johnson WH, Ausubel FM (1985) Nitrogen fixation specific regulatory genes ofKlebsiella pneumoniae andRhizobium meliloti share homology with the general nitrogen regulatory genentrC ofKlebsiella pneumoniae. Nucleic Acids Res 13:4539–4555

    Google Scholar 

  • Buikema WJ, Klingensmith JA, Gibbons SL, Ausubel FM (1987) Conservation of structure and location ofRhizobium meliloti andKlebsiella pneumoniae nifB genes. J Bacteriol 169:1120–1126

    Google Scholar 

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

    Google Scholar 

  • Ebeling S, Noti JD, Hennecke H (1988) Identification of a newBradyrhizobium japonicum gene (frxA) encoding a ferredoxin-like protein. J Bacteriol 170:1999–2001

    Google Scholar 

  • George DG, Hunt LT, Yeh L-SL, Barker WC (1985) New perspectives on bacterial ferredoxin evolution. J Mol Evol 22:20–31

    Google Scholar 

  • Grönger P, Priefer UB, Klipp W, Pühler A (1988) TheRhizobium leguminosarum nifA, nifB gene region can be used for complementation ofRhizobium meliloti mutants. In: Bothe H, de Bruijn FM, Newton WE (eds) Nitrogen Fixation: Hundred Years After. Gustav Fischer Stuttgart New York, p 367

    Google Scholar 

  • Hedges RW, Jacob AC (1974) Transposition of ampicillin resistance from RP4 to other replicons. Mol Gen Genet 132:31–40

    Google Scholar 

  • Hirsch PR, Wang CL, Woodward MJ (1986) Construction of a Tn5 derivative determining resistance to gentamicin and spectinomycin using a fragment cloned from R1033. Gene 48:203–209

    Google Scholar 

  • Hynes MF, Simon R, Müller P, Niehaus K, Labes M, Pühler A (1986) The two megaplasmids ofRhizobium meliloti are involved in the effective nodulation of alfalfa. Mol Gen Genet 202:356–362

    Google Scholar 

  • Joerger RD, Bishop PE (1988) Nucleotide sequence and genetic analysis of thenifB-nifQ region fromAzotobacter vinelandii. J Bacteriol 170:1475–1487

    Google Scholar 

  • Jouanneau Y, Meyer C, Gaillard J, Vignais PM (1990) Purification and characterization of a 7Fe-ferredoxin fromRhodobacter capsulatus. Biochem Biophys Res Commun 171:273–279

    Google Scholar 

  • Klipp W (1990) Organization and regulation of nitrogen fixation genes inRhodobacter capsulatus. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: Achievements and objectives. Chapman and Hall New York London, pp 467–474

    Google Scholar 

  • Klipp W, Reiländer H, Schlüter A, Krey R, Pühler A (1989) TheRhizobium meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed withnifA andnifB. Mol Gen Genet 216:293–302

    Google Scholar 

  • Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: A laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Martin AE, Burgess BK, Stout CD, Cash VL, Dean DR, Jensen GM (1990) Site-directed mutagenesis ofAzotobacter vinelandii ferredoxin I: [Fe-S] cluster-driven protein rearrangement. Proc Natl Acad Sci USA 87:598–602

    Google Scholar 

  • Matteucci MD, Caruthers MH (1981) Synthesis of deoxynucleotides on a polymer support. J Am Chem Soc 102:3185–3191

    Google Scholar 

  • Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York

    Google Scholar 

  • Moreno-Vivian C, Hennecke S, PÜhler A, Klipp W (1989) Open reading frame 5 (ORF5), encoding a ferredoxin-like protein, andnifQ are cotranscribed withnifE, nifty, nifX, and ORF4 inRhodobacter capsulatus. J Bacteriol 171:2591–2598

    Google Scholar 

  • Mulligan ME, Buikema WJ, Haselkorn R (1988) Bacterial-type ferredoxin genes in the nitrogen fixation regions of the cyanobacteriumAnabaena sp. strain PCC 7120 andRhizobium meliloti. J Bacteriol 170:4406–4410

    Google Scholar 

  • Robson R, Woodley P, Jones R (1986) Second gene (nifH *) coding for a nitrogenase iron protein inAzotobacter chroococcum is adjacent to a gene coding for a ferredoxin-like protein. EMBO J 5:1159–1163

    Google Scholar 

  • Ruvkun GB, Ausubel FM (1980) Interspecies homology of nitrogenase genes. Proc Natl Acad Sci USA 77:191–195

    Google Scholar 

  • Saeki K, Miyatake Y, Young DA, Marrs BL, Matsubara H (1990) A plant ferredoxin-like gene is located upstream of ferredoxin I gene (fdxN) ofRhodobacter capsulatus. Nucleic Acids Res 18:1060

    Google Scholar 

  • Schatt E, Jouanneau Y, Vignais P (1989) Molecular cloning and sequence analysis of the structural gene of ferredoxin I from the photosynthetic bacteriumRhodobacter capsulatus. J Bacteriol 171:6218–6226

    Google Scholar 

  • Schmehl M, Meyer zu Vilsendorf A, Hennecke S, Masepohl B, Klipp W (1990) DNA sequence and mutational analysis of three operons ofRhodobacter capsulatus nif region A containing genes encoding ferredoxin-like proteins. In: Gresshoff PM, Roth LE, Stacey G, Newton WE (eds) Nitrogen fixation: Achievements and objectives. Chapman and Hall New York London, p 580

    Google Scholar 

  • Schomburg D, Reichelt J (1988) BRAGI: A comprehensive protein modelling program system. J Mol Graphics 4:161–165

    Google Scholar 

  • Shapira SK, Chou J, Richard FV, Casadaban MJ (1983) New versatile plasmid vectors for expression of hybrid proteins coded by a cloned gene fused tolacZ gene sequences encoding an enzymatically active carboxy-terminal portion of β-galactosidase. Gene 25:71–82

    Google Scholar 

  • Simon R, Priefer U, Pühler A (1983) A broad host range mobilization system forin vivo genetic engineering: Transposon mutagenesis in gram negative bacteria. Biotechnology 1:784–791

    Google Scholar 

  • Stout CD (1989) Refinement of the 7Fe ferredoxin fromAzotobacter uinelandii at 1.9A resolution. J Mol Biol 205:545–555

    Google Scholar 

  • Szeto WW, Zimmermann JL, Sundaresan V, Ausubel FM (1984) ARhizobium meliloti symbiotic regulatory gene. Cell 36:1035–1043

    Google Scholar 

  • Vieira J, Messing J (1982) The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259–268

    Google Scholar 

  • Weber G, Reiländer H, Pühler A (1985) Mapping and expression of a regulatory nitrogen fixation gene (fixD) ofRhizobium meliloti. EMBO J 4:2751–2756

    Google Scholar 

  • Yates MG, O'Donnell MJ, Lowe DJ, Bothe H (1978) Ferredoxins from nitrogen-fixing bacteria. Eur J Biochem 85:291–299

    Google Scholar 

  • Yoch DC, Arnon DI (1975) Comparison of two ferredoxins fromRhodospirillum rubrum as electron carriers for the native nitrogenase. J Bacteriol 121:743–745

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Lehrstuhl für Genetik, Fakultät für Biologie, Universität Bielefeld, Postfach 8640, W-4800, Bielefeld 1, Germany

    Bernd Masepohl, Michael Kutsche, Kai-Uwe Riedel, Manfred Schmehl, Werner Klipp & Alfred Pühler

Authors
  1. Bernd Masepohl
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Michael Kutsche
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Kai-Uwe Riedel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Manfred Schmehl
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Werner Klipp
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Alfred Pühler
    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

Masepohl, B., Kutsche, M., Riedel, KU. et al. Functional analysis of the cysteine motifs in the ferredoxin-like protein FdxN ofRhizobium meliloti involved in symbiotic nitrogen fixation. Molec. Gen. Genet. 233, 33–41 (1992). https://doi.org/10.1007/BF00587558

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00587558

Key words

Search

Navigation