Molecular and General Genetics MGG

, Volume 218, Issue 2, pp 340–347

Genes coding for the reversible ADP-ribosylation system of dinitrogenase reductase from Rhodospirillum rubrum

  • Wayne P. Fitzmaurice
  • Leonard L. Saari
  • Robert G. Lowery
  • Paul W. Ludden
  • Gary P. Roberts


Nitrogen fixation activity in the photosynthetic bacterium Rhodospirillum rubrum is controlled by the reversible ADP-ribosylation of the dinitrogenase reductase component of the nitrogenase enzyme complex. This report describes the cloning and characterization of the genes encoding the ADP-ribosyltransferase (draT) and the ADP-ribosylglycohydrolase (draG) involved in this regulation. These genes are shown to be contiguous on the R. rubrum chromosome and highly linked to the nifHDK genes. Sequence analysis revealed the use of TTG as the initiation codon of the draT gene as well as a potential open reading frame immediately downstream of draG. The mono-ADP-ribosylation system in R. rubrum is the first in which both the target protein and modifying enzymes as well as their structural genes have been isolated, making it the model system of choice for analysis of this post-translational regulatory mechanism.

Key words

Nitrogen fixation draT draG TTG initiation codon 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Bérard J, Bélanger G, Corriveau P, Gingras G (1986) Molecular cloning and sequence of the B880 holochrome gene from Rhodospirillum rubrum. J Biol Chem 261:82–87Google Scholar
  2. Berent SL, Mahmoudi M, Torczynski RM, Bragg PW, Bollon AP (1985) Comparison of oligonucleotide and long DNA fragments as probes in DNA and RNA dot, Southern, Northern, colony and plaque hybridizations. Bio Techniques 3:208–220Google Scholar
  3. Berg D, Weiss A, Crossland L (1980) The polarity of Tn5 insertion mutations in Escherichia coli. J Bacteriol 142:439–446Google Scholar
  4. Birnboim HC (1983) A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol 100:243–255Google Scholar
  5. Bolivar F, Rodriguez RL, Green PJ, Betlach MC, Heynecker HL, Boyer HW, Crosa JH, Falkow S (1977) Construction and characterization of new cloning vehicles II. A multipurpose cloning system. Gene 2:95–113Google Scholar
  6. Bose SK, Gest H, Ormerod JG (1961) Light-activated hydrogenase activity in a photosynthetic bacterium: a permeability phenomenon. J Biol Chem 236:PC13-PC14Google Scholar
  7. Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in Escherichia coli. J Mol Biol 41:459–472Google Scholar
  8. Clewell DB, Helinski DR (1969) Supercoiled circular DNA-protein complex in Escherichia coli: purification and induced conversion to an open circular DNA form. Proc Natl Acad Sci USA 62:1159–1166Google Scholar
  9. de Bruijn FJ, Lupinski JR (1984) The use of transposon Tn5 mutagenesis in the rapid generation of correlated physical and genetic maps of DNA segments cloned into multicopy plasmids — a review. Gene 27:131–149Google Scholar
  10. Devereux J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12:387–395Google Scholar
  11. Ditta G (1986) Tn5 mapping of Rhizobium nitrogen fixation genes. Methods Enzymol 118:519–528Google Scholar
  12. Falk G, Hampe A, Walker JE (1985) Nucleotide sequence of the Rhodospirillum rubrum atp operon. Biochem J 228:391–407Google Scholar
  13. Feinberg AP, Vogelstein B (1984) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem 137:266–267Google Scholar
  14. Fu H-A, Fitzmaurice WP, Lehman LJ, Roberts GP, Burris RH (1988) Regulation of nitrogenase activity in azospirilla, herbaspirilla and acetobacter: and cloning of draG-and draT-homologous genes of A. lipoferum SpBr17. In: Bothe H, de Bruijn FL, Newton WE (eds) Nitrogen Fixation: Hundred Years After. Gustav Fischer, Stuttgart, p 336Google Scholar
  15. Gribskov M, Devereux J, Burgess RR (1984) The codon preference plot: graphic analysis of protein coding sequences and prediction of gene expression. Nucleic Acids Res 12:539–549Google Scholar
  16. Hartman A, Fu H, Burris RH (1986) Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp. J Bacteriol 165:864–870Google Scholar
  17. Kanemoto RH, Ludden PW (1984) Effect of ammonia, darkness, and phenazine methosulfate on whole-cell nitrogenase activity and Fe protein modification in Rhodospirillum rubrum. J Bacteriol 158:713–720Google Scholar
  18. Kush A, Elmerich C, Aubert JP (1985) Nitrogenase of Sesbania Rhizobium strain ORS571: purification, properties and ‘switchoff’ by ammonia. J Gen Microbiol 131:1765–1777Google Scholar
  19. Lehman LJ, Fitzmaurice WP, Roberts GP (1988) The cloning and mutagenesis of nifHDK of Rhodospirillum rubrum. In: Bothe H, de Bruijin FJ, Newton WE (eds) Nitrogen Fixation: Hundred Years After. Gustav Fischer, Stuttgart, p 174Google Scholar
  20. Lowery RG, Saari LL, Ludden PW (1986) Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro. J Bacteriol 166:513–518Google Scholar
  21. Ludden PW, Roberts GP (1988) Regulation of nitrogenase activity by reversible ADP-ribosylation. Curr Top Cell Regul (in press)Google Scholar
  22. Ludden PW, Okon Y, Burris RH (1988) The nitrogenase system of Spirillum lipoferum. Biochem J 173:1001–1003Google Scholar
  23. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  24. Moss J, Vaughan M (1977) Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor. J Biol Chem 252:2455–2457Google Scholar
  25. Moss J, Jacobson MK, Stanley SJ (1985) Reversibility of argininespecific mono(ADP-ribosyl)ation: identification in erythrocytes of an ADP-ribose-cl-arginine cleavage enzyme. Proc Natl Acad Sci USA 82:5603–5607Google Scholar
  26. Nargang F, McIntosh L, Somerville C (1984) Nucleotide sequence of the ribulosebisphosphate carboxylase gene from Rhodospirillum rubrum. Mol Gen Genet 193:220–224Google Scholar
  27. Pope MR, Saari LL, Ludden PW (1986) N-glycohydrolysis of adenosine diphosphoribosyl arginine linkages by dinitrogenase reductase activating glycohydrolase (activating enzyme) from Rhodospirillum rubrum. J Biol Chem 261:10104–10111Google Scholar
  28. Reddy P, Peterkofsky A, McKenny K (1985) Translational efficiency of the Escherichia coli adenylate cyclase gene: Mutating the UUG initiation codon to GUG or AUG results in increased gene expression. Proc Natl Acad Sci USA 82:5656–5660Google Scholar
  29. Reed KC, Mann DA (1985) Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res 13:7207–7221Google Scholar
  30. Saari LL, Triplett EW, Ludden PW (1984) Purification and properties of the activating enzyme for iron protein of nitrogenase from the photosynthetic bacterium Rhodospirillum rubrum. J Biol Chem 259:15502–15508Google Scholar
  31. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467Google Scholar
  32. Shine J, Dalgarno L (1974) The 3′-terminal sequence of E. coli 16S ribosomal RNA: complementarity to nonsense triplets and ribosome binding sites. Proc Natl Acad Sci USA 71:1342–1346Google Scholar
  33. Skórko R, Zillig W, Rohrer H, Fujiki H, Mailhammer R (1977) Purification and properties of the NAD+: protein ADP-ribosyltransferase responsible for the T4-phage-induced modification of the β subunit of DNA-dependent RNA polymerase of Escherichia coli. Eur J Biochem 79:55–66Google Scholar
  34. Southern EM (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 98:503–517Google Scholar
  35. Stormo GD, Schneider TD, Gold LM (1982) Characterization of translational initiation sites in E. coli. Nucleic Acids Res 10:2971–2996Google Scholar
  36. Tanuma S, Yagi T, Johnson G (1985) Endogenous ADP-ribosylation of high mobility group proteins 1 and 2 and histone HI following DNA damage in intact cells. Arch Biochem Biophys 237:38–42Google Scholar
  37. Van Ness BG, Howard JB, Bodley JW (1978) Isolation and properties of the trypsin-derived ADP-ribosyl peptide from diphtheria toxin-modified yeast elongation factor 2. J Biol Chem 253:8687–8690Google Scholar
  38. Watkins PA, Yost DA, Chang AW, Mekalanos JJ, Moss J (1985) Detection of NAD: arginine ADPribosyltransferases in animal tissues using 125I-labeled 1-(p-hydroxyphenyl) 2-guanidinoethane as ADPribose acceptor. Biochim Biophys Acta 840:401–408Google Scholar
  39. West RE Jr, Moss J, Vaughan M, Liu T, Liu T-Y (1985) Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose aceptor site. J Biol Chem 260:14428–14430Google Scholar
  40. Zumft WG, Castillo F (1978) Regulatory properties of the nitrogenase from Rhodopseudomonas palustris. Arch Microbiol 117:53–60Google Scholar

Copyright information

© Springer-Verlag 1989

Authors and Affiliations

  • Wayne P. Fitzmaurice
    • 1
  • Leonard L. Saari
    • 2
  • Robert G. Lowery
    • 2
  • Paul W. Ludden
    • 2
  • Gary P. Roberts
    • 1
  1. 1.Department of BacteriologyUniversity of WisconsinMadisonUSA
  2. 2.Department of BiochemistryUniversity of WisconsinMadisonUSA
  3. 3.Department of GeneticsNorth Carolina State UniversityRaleighUSA
  4. 4.Agricultural Chemicals DepartmentE.I. Du Pont de Nemous and Co.WilmingtonUSA
  5. 5.Department of BiologyUniversity of UtahSalt Lake CityUSA

Personalised recommendations