Posttranslational modification of dinitrogenase reductase in Rhodospirillum rubrum treated with fluoroacetate

  • Natalia AkentievaEmail author
Original Paper


Nitrogen fixation is one of the major biogeochemical contributions carried out by diazotrophic microorganisms. The goal of this research is study of posttranslational modification of dinitrogenase reductase (Fe protein), the involvement of malate and pyruvate in generation of reductant in Rhodospirillum rubrum. A procedure for the isolation of the Fe protein from cell extracts was developed and used to monitor the modification of the Fe protein in vivo. The subunit pattern of the isolated the Fe protein after sodium dodecyl sulfate–polyacrylamide gel electrophoresis was assayed by Western blot analysis. Whole-cell nitrogenase activity was also monitored during the Fe protein modification by gas chromatograpy, using the acetylene reduction assay. It has been shown, that the addition of fluoroacetate, ammonia and darkness resulted in the loss of whole-cell nitrogenase activity and the in vivo modification of the Fe protein. For fluoroacetate, ammonia and darkness, the rate of loss of nitrogenase activity was similar to that for the Fe protein modification. The addition of NADH and reillumination of a culture incubated in the dark resulted in the rapid restoration of nitrogenase activity and the demodification of the Fe protein. Fluoroacetate inhibited the nitrogenase activity of R. rubrum and resulted in the modification of the Fe protein in cells, grown on pyruvate or malate as the endogeneous electron source. The nitrogenase activity in draTG mutant (lacking DRAT/DRAG system) decreased after the addition of fluoroacetate, but the Fe protein remained completely unmodified. The results showed that the reduced state of cell, posttranslational modifications of the Fe protein and the DRAT/DRAG system are important for nitrogenase activity and the regulation of nitrogen fixation.


Dinitrogenase reductase (Fe protein) Nitrogen fixation Posttranslational modification Rhodospirillum rubrum 



Authors would like to thank the financial support of FASE, State Registration of Research Study Work is # 01201361874.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict interests.


  1. Brostedt E, Lindblad A, Jansson J, Nordlund S (1997) Electron transport to nitrogenase in Rhodospirillum rubrum: the role of NAD(P)H as electron donor and the effect of fluoroacetate on nitrogenase activity. FEMS Microbiol Lett 150:263–267CrossRefGoogle Scholar
  2. Burris RH (1991) Nitrogenases J Biol Chem 266:9339–9342PubMedGoogle Scholar
  3. Eisenberg MA (1953) The tricarboxylic acid cycle in Rhodospirillum rubrum. J Biol Chem 203:815–836PubMedGoogle Scholar
  4. Elsden SR, Ormerod JG (1956) The effect of fluoroacetate on the metabolism of Rhodospirillum rubrum. Biochem J 63:691–701CrossRefPubMedPubMedCentralGoogle Scholar
  5. Gest H, Ormerod JG, Ormerod KS (1962) Photometabolism of Rhodospirillum rubrum: light-dependent dissimilation of organic compounds to carbon dioxide and molecular hydrogen by an anaerobic citric acid cycle. Arch Biochem Biophys 97:21–33CrossRefPubMedGoogle Scholar
  6. Gorell TE, Uffen RL (1978) Reduction of nucleotide adenine dinucleotide by pyruvate: lipoate oxidoreducatse in anaerobic, dark grown Rhodospirillum rubrum mutant C. J Bacteriol 134:830–836Google Scholar
  7. Grunwald SK, Ryle MJ, Lanzilotta WN, Ludden PW (2000) ADP-ribosylation of variants of Azotobacter vinelandii dinitrogenase reductase by Rhodospirillum rubrum dinitrogenase reductase ADP-ribosyltransferase. J Bacteriol 182:2597–2603CrossRefPubMedPubMedCentralGoogle Scholar
  8. Halbleib CM, Zhang, Yaoping, Ludden PW (2000) Regulation of dinitrogenase reductase ADP-ribosyltransferase and dinirogenase reductase-activating glycohydrolase by a redox-dependent conformational change of nitrogenase Fe protein. J Biol Chem 275:3493–3500CrossRefPubMedGoogle Scholar
  9. Heinrich D, Raberg M, Fricke P, Kenny ST, Morales-Gamez L, Babu RP, O’Connor KE, Steinbüchel A (2016) Syngas-derived medium-chain-length PHA synthesis in engineered Rhodospirillum rubrum. Appl Environ Microbiol 82(20):6132–6140CrossRefPubMedPubMedCentralGoogle Scholar
  10. Huergo LF, Merrick M, Pedrosa FO, Chubatsu LS, Araujo LM, Souza EM (2007) Ternary complex formation between AmtB, GlnZ and the nitrogenase regulatory enzyme DraG reveals a novel facet of nitrogen regulation in bacteria. Mol Microbiol 66:1523–1535PubMedGoogle Scholar
  11. Jackson JB, Crofts AR (1968) Energy-linked reduction of nicotinamide adenine dinucleotides in cells of Rhodospirillum rubrum. Biochem Biophys Res Commun 32:908–915CrossRefPubMedGoogle Scholar
  12. Javelle A, Lupo D, Ripoche P, Fulford T, Merrick M, Winkler FK (2008) Substrate binding, deprotonation, and selectivity at the periplasmic entrance of the Escherichia coli ammonia channel AmtB. Proc Natl Acad Sci USA 105:5040–5045CrossRefPubMedGoogle Scholar
  13. Jonsson A, Teixeira PF, Nordlund S (2007) The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by alpha-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro. FEBS J 274 (10):2449–2460CrossRefPubMedGoogle Scholar
  14. 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 158:713–720PubMedPubMedCentralGoogle Scholar
  15. Kanemoto RH, Ludden PW (1987) Amino acid concentrations in Rhodospirillum rubrum during expression and switch-off of nitrogenase activity. J Bacteriol 169:3035–3043CrossRefPubMedPubMedCentralGoogle Scholar
  16. Koch B, Evans HJ (1966) Reduction of acetylene to ethylene by soybean root nodules. Plant Physiol 41:1748–1750CrossRefPubMedPubMedCentralGoogle Scholar
  17. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefPubMedPubMedCentralGoogle Scholar
  18. Li J-D, Hu C-Z, Yoch DC (1987) Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by ammonium or darkness. J Bacteriol 169:231–237CrossRefPubMedPubMedCentralGoogle Scholar
  19. Liang JH, Nielsen GM, Lies DP, Burris RH, Roberts GP, Ludden PW (1991) Mutations in the draT and draG genes of Rhodospirillum rubrum result in loss of regulation of nitrogenase by reversible ADP-ribosylation. J Bacteriol 173:6903–6909CrossRefPubMedPubMedCentralGoogle Scholar
  20. Lowery RG, Ludden PW (1988) Purification and properties of dinitrogenase reductase ADPribosyltransferase from the photosynthetic bacterium Rhodospirillum rubnum. J Biol Chem 263:16714–16719PubMedGoogle Scholar
  21. Lowery RG, Ludden PW (1989) Effect of nucleotides on the activity of dinitrogenase reductase ADPribosyltransferasefrom Rhodospirillum rubrum. Biochemistry 28:4956–4961CrossRefPubMedGoogle Scholar
  22. Lowery RG, Saari LL, Ludden PW (1986) Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro. J Bacteriol 166:513–518CrossRefPubMedPubMedCentralGoogle Scholar
  23. Ludden PW, Burries RH (1976) Activating factor for the iron protein of nitrogenase from Rhodospirillum rubrum. Science 194:424–426CrossRefPubMedGoogle Scholar
  24. Ludden PW, Burris RH (1978) Purification and properties of nitrogenase from Rhodospirillum rubrum, and evidence for phosphate, ribose and an adenine-like unit covalently bound to the iron protein. Biochem J 175:251–259CrossRefPubMedPubMedCentralGoogle Scholar
  25. Luderitz R, Klemme JH (1977) Isolation and characterization of a membrane bound pyruvate dehydrogenase complex from the phototrophic bacterium Rhodospirillum rubrum. Z Naturforsch 32c:351–361CrossRefGoogle Scholar
  26. Moure VR, Danyal K, Yang ZY, Wendroth S, Müller-Santos M, Pedrosa FO, Scarduelli M, Gerhardt EC, Huergo LF, Souza EM, Seefeldt LC (2012) The nitrogenase regulatory enzyme dinitrogenase reductase ADP-ribosyltransferase (DraT) is activated by direct interaction with the signal transduction protein GlnB. J Bacteriol 195:279–286. CrossRefPubMedGoogle Scholar
  27. Nordlund S, Hogbom M (2013) ADP-ribosylation, a mechanism regulating nitrogenase activity. FEBS J 280:3484–3490CrossRefPubMedGoogle Scholar
  28. Nordlund S, Högland L (1986) Studies of the adenylate and pyridine nucleotide pools during nitrogenase switch-off in Rhodospirillum rubrum. Plant Soil 90:203–209CrossRefGoogle Scholar
  29. Nordlund S, Noren A (1984) Dependence on divalent cations of the activation of inactive Fe-protein of nitrogenase from Rhodospirillum rubrum. Biochim Biophys Acta 791:21–27CrossRefGoogle Scholar
  30. Noren A, Nordlund S (1994) Changes in the NAD (P) H concentration caused by addition of nitrogenase switch-off effectors in Rhodospirillum rubrum G-9, as measured by fluorescence. FEBS Lett 356:43–45CrossRefPubMedGoogle Scholar
  31. Noren A, Soliman A, Nordlund S (1997) The role of NAD+ as a signal during nitrogenase switch-off in Rhodospirillum rubrum. Biochem J 322:829–832CrossRefPubMedPubMedCentralGoogle Scholar
  32. O’Neal L, Ryu MH, Gomelsky M, Alexandre G (2017) Optogenetic manipulation of cyclic di-GMP (c-di-GMP) levels reveals the role of c-di-GMP in regulating aerotaxis receptor activity in Azospirillum brasilense. J Bacteriol 22:199–218Google Scholar
  33. Ormerod JG, Ormerod KS, Gest H (1961) Light-dependent utilization of organic compounds and photoproduction of molecular hydrogen by photosynthetic bacteria; relationships with nitrogen metabolism. Arch Biochem Biophys 94:449–463CrossRefPubMedGoogle Scholar
  34. Paul TD, Ludden PW (1984) Adenine nucleotide level in Rhodospirillum rubrum during switch-off of nitrogenase activity. Biochem J 224:961–969CrossRefPubMedPubMedCentralGoogle Scholar
  35. Ponnuraj RK, Rubio LM, Grunwald SK, Ludden PW (2005) NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii. FEBS Lett 579:5751–5758CrossRefPubMedGoogle Scholar
  36. Pope MR, Murrell SA, Ludden PW (1985) Covalent modification of the iron protein of nitrogenase from Rhodospirillum rubrum by adenosine diphosphoribosylation of a specific arginine residue. Proc Natl Acad Sci USA 82(10):3173–3177CrossRefPubMedGoogle Scholar
  37. Pratt DC, Frenkel AW (1959) Studies on nitrogen fixation and photosynthesis of Rhodospirillum rubrum. Plant Physiol 34:333–337CrossRefPubMedPubMedCentralGoogle Scholar
  38. Rabouille S, Van de Waal DB, Matthijs HC, Huisman J (2014) Nitrogen fixation and respiratory electron transport in the cyanobacterium Cyanothece under different light/dark cycles. FEMS Microbiol Ecol 87:630–638CrossRefPubMedGoogle Scholar
  39. 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 258:12064–12068Google Scholar
  40. Schick IHJ (1971) Regulation of photoreduction in Rhodospirillum rubrum by ammonia. Arch Mikrobiol 75:110–120CrossRefPubMedGoogle Scholar
  41. Seefeldt LC, Hoffman BH, Dean DR (2009) Mechanism of Mo-dependent nitrogenase. Annu Rev Biochem 78:701–722CrossRefPubMedPubMedCentralGoogle Scholar
  42. Selao TT, Nordlund S, Norén A (2008) Comparative proteomic studies in Rhodospirillum rubrum grown under different nitrogen conditions. J Proteome Res 7:3267–3275CrossRefPubMedGoogle Scholar
  43. Selao TT, Edgren T, Wang H, Noren A, Nordlund S (2011) Effect of pyruvate on the metabolic regulation of nitrogenase activity in Rhodospirillum rubrum in darkness. Microbiology 157:1834–1840CrossRefPubMedGoogle Scholar
  44. Soliman A, Nordlund S (1992) Studies on the effect of NAD (H) on nitrogenase activity in Rhodospirillum rubrum. Arch Microbiol 157:431–435CrossRefPubMedGoogle Scholar
  45. Stewart WDP, Fitzgerald GP, Burris RH (1967) In situ studies on N2 fixation using the acetylene reduction technique. Proc Natl Acad Sci USA 58:2071–2078CrossRefPubMedGoogle Scholar
  46. Teixeira PF, Jonsson A, Frank M, Wang H, Nordlund S (2008) Interaction of the signal transduction protein GlnJ with the cellular targets AmtB1, GlnE and GlnD in Rhodospirillum rubrum: dependence on manganese, 2-oxoglutarate and the ADP/ATP ratio. Microbiology 154(Pt 8):2336–2347CrossRefPubMedGoogle Scholar
  47. Teixeira PF, Wang H, Nordlund S (2010) Nitrogenase switch-off and regulation of ammonium assimilation in response to light deprivation in Rhodospirillum rubrum are influenced by the nitrogen source used during growth. Journal of bacteriology 192:1463–1466CrossRefPubMedGoogle Scholar
  48. Tortajada M (2017) New waves underneath the purple strain. Microb Biotechnol 10(6):1297–1299CrossRefPubMedGoogle Scholar
  49. Wolfe DM, Zhang Y, Roberts GP (2007) Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum. J Bacteriol 189:6861–6869CrossRefPubMedPubMedCentralGoogle Scholar
  50. Yoch DC (1979) Manganese, an essential trace element for N2 fixation by Rhodospirillum rubrum and Rhodopseudomonas capsulata: role in nitrogenase regulation. J Bacteriol 140:987–995PubMedPubMedCentralGoogle Scholar

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© Springer Nature B.V. 2018

Authors and Affiliations

  1. 1.Department of Kinetics of Chemical and Biological Processes, Institute of Problems of Chemical PhysicsRussian Academy of SciencesChernogolovkaRussia

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