Nitrogen and Molybdenum Control of Nitrogen Fixation in the Phototrophic Bacterium Rhodobacter capsulatus

Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 675)


The vast majority of the purple nonsulfur photosynthetic bacteria are diazotrophs, but the details of the complex regulation of the nitrogen fixation process are well understood only for a few species. Here we review what is known of the well-studied Rhodobacter capsulatus, which contains two different nitrogenases, a standard Mo-nitrogenase and an alternative Fe-nitrogenase, and which has overlapping transcriptional control mechanisms with regard to the presence of fixed nitrogen, oxygen, and molybdenum as well as the capability for the post-translational control of both nitrogenases in response to ammonium. R. capsulatus has two PII proteins, GlnB and GlnK, which play key roles in nitrogenase regulation at each of three different levels: activation of transcription of the nif-specific activator NifA, the post-translational control of NifA activity, and the regulation of nitrogenase activity through either ADP-ribosylation of NifH or an ADP-ribosylation-independent pathway. We also review recent work that has led to a detailed characterization of the molybdenum transport and regulatory system in R. capsulatus that ensures activity of the Mo-nitrogenase and repression of the Fe-nitrogenase, down to extremely low levels of molybdenum.


Nitrogen Fixation Rhodobacter Capsulatus Rhodopseudomonas Palustris MoFe Protein Purple Nonsulfur Bacterium 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



Research in the authors’ laboratories is supported by Deutsche Forschungsgemeinschaft (BM) and the Natural Sciences and Engineering Research Council of Canada Discovery Grants Program (PCH).


  1. Andrade SL, Einsle O (2007) The Amt/Mep/Rh family of ammonium transport proteins. Mol Membr Biol 24:357–365PubMedCrossRefGoogle Scholar
  2. Boeckstaens M, André B, Marini AM (2008) Distinct transport mechanisms in yeast ammonium transport/sensor proteins of the Mep/Amt/Rh family and impact on filamentation. J Biol Chem 283:21362–21370PubMedCrossRefGoogle Scholar
  3. Bowman WC, Kranz RG (1998) A bacterial ATP-dependent, enhancer binding protein that activates the housekeeping RNA polymerase. Genes Dev 12:1884–1893PubMedCrossRefGoogle Scholar
  4. Cullen PJ, Bowman WC, Hartnett DF, Reilly SC, Kranz RG (1998) Translational activation by an NtrC enhancer-binding protein. J Mol Biol 278:903–914PubMedCrossRefGoogle Scholar
  5. Cullen PJ, Bowman WC, Kranz RG (1996) In vitro reconstitution and characterization of the Rhodobacter capsulatus NtrB and NtrC two-component system. J Biol Chem 271:6530–6536PubMedCrossRefGoogle Scholar
  6. Cullen PJ, Foster-Hartnett D, Gabbert KK, Kranz RG (1994) Structure and expression of the alternative sigma factor, RpoN, in Rhodobacter capsulatus; Physiological relevance of an autoactivated nifU2-rpoN superoperon. Mol Microbiol 11:51–65PubMedCrossRefGoogle Scholar
  7. Dixon R, Kahn D (2004) Genetic regulation of biological nitrogen fixation. Nat Rev 2:621–631CrossRefGoogle Scholar
  8. Drepper T, Groß S, Yakunin AF, Hallenbeck PC, Masepohl B, Klipp W (2003) Role of GlnB and GlnK in ammonium control of both nitrogenase systems in the phototrophic bacterium Rhodobacter capsulatus. Microbiol 149:2203–2212CrossRefGoogle Scholar
  9. Fischer H-M (1994) Genetic regulation of nitrogen fixation in rhizobia. Microbiol Rev 58:352–386PubMedGoogle Scholar
  10. Fischer H-M, Bruderer T, Hennecke H (1988) Essential and non-essential domains in the Bradyrhizobium japonicum NifA protein: Identification of indispensable cysteine residues potentially involved in redox reactivity and/or metal binding. Nucleic Acids Res 16: 2207–2224PubMedCrossRefGoogle Scholar
  11. Fong RN, Kim KS, Yoshihara C, Inwood WB, Kustu S (2007) The W148L substitution in the Escherichia coli ammonium channel AmtB increases flux and indicates that the substrate is an ion. Proc Natl Acad Sci USA 104:18706–18711PubMedCrossRefGoogle Scholar
  12. Forchhammer K (2008) PII signal transducers: Novel functional and structural insights. Trends Microbiol 16:65–72PubMedCrossRefGoogle Scholar
  13. Foster-Hartnett D, Cullen PJ, Monika EM, Kranz RG (1994) A new type of NtrC transcriptional activator. J Bacteriol 176:6175–6187PubMedGoogle Scholar
  14. Foster-Hartnett D, Kranz RG (1992) Analysis of the promoters and upstream sequences of nifA1 and nifA2 in Rhodobacter capsulatus; activation requires ntrC but not rpoN. Mol Microbiol 6:1049–1060PubMedCrossRefGoogle Scholar
  15. Glazer AN, Kechris KJ (2009) Conserved amino acid sequence features in the α subunits of MoFe, VFe, and FeFe nitrogenases. PLoS ONE 4:e6136Google Scholar
  16. Gourley DG, Schüttelkopf AW, Anderson LA, Price NC, Boxer DH, Hunter WN (2001) Oxyanion binding alters conformation and quaternary structure of the C-terminal domain of the transcriptional regulator ModE. Implications for molybdate-dependent regulation, signaling, storage, and transport. J Biol Chem 276:20641–20647PubMedCrossRefGoogle Scholar
  17. Grunden AM, Shanmugam KT (1997) Molybdate transport and regulation in bacteria. Arch Microbiol 168:345–354PubMedCrossRefGoogle Scholar
  18. Hall DR, Gourley DG, Leonard GA, Duke EM, Anderson LA, Boxer DH, Hunter WN (1999) The high-resolution crystal structure of the molybdate-dependent transcriptional regulator (ModE) from Escherichia coli: A novel combination of domain folds. EMBO J 18:1435–1446PubMedCrossRefGoogle Scholar
  19. Hu Y, Fay AW, Lee CC, Yoshizawa J, Ribbe MW (2008) Assembly of nitrogenase MoFe protein. Biochemistry 47:3973–3981PubMedCrossRefGoogle Scholar
  20. Hübner P, Masepohl B, Klipp W, Bickle TA (1993) nif gene expression studies in Rhodobacter capsulatus: ntrC-independent repression by high ammonium concentrations. Mol Microbiol 10:123–132PubMedCrossRefGoogle Scholar
  21. 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
  22. Javelle A, Merrick M (2005) Complex formation between AmtB and GlnK: An ancestral role in prokaryotic nitrogen control. Biochem Soc Trans 33:170–172PubMedCrossRefGoogle Scholar
  23. Jiang P, Peliska JA, Ninfa AJ (1998a) Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC of Escherichia coli and its interaction with PII protein. Biochemistry 37:12782–12794PubMedCrossRefGoogle Scholar
  24. Jiang P, Peliska JA, Ninfa AJ (1998b) Reconstitution of the signal-transduction bicyclic cascade responsible for the regulation of Ntr gene transcription in Escherichia coli. Biochemistry 37:12795–12801PubMedCrossRefGoogle Scholar
  25. Jiang P, Peliska JA, Ninfa AJ (1998c) The regulation of Escherichia coli glutamine synthetase revisited: Role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state. Biochemistry 37:12802–12810PubMedCrossRefGoogle Scholar
  26. Joshi HM, Tabita FR (1996) A global two component signal transduction system that integrates the control of photosynthesis, carbon dioxide assimilation, and nitrogen fixation. Proc Natl Acad Sci USA 93:14515–14520PubMedCrossRefGoogle Scholar
  27. Kutsche M, Leimkühler S, Angermüller S, Klipp W (1996) Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of σ54, and repressed by molybdenum. J Bacteriol 178: 2010–2017PubMedGoogle Scholar
  28. Lehman LJ, Roberts GP (1991) Identification of an alternative nitrogenase system in Rhodospirillum rubrum. J Bacteriol 173:5705–5711PubMedGoogle Scholar
  29. Lim S-K, Kim SJ, Cha SH, Oh Y-K, Rhee H-J, Kim M-S, Lee JK (2009) Complete genome sequence of Rhodobacter sphaeroides KD131. J Bacteriol 191:1118–1119PubMedCrossRefGoogle Scholar
  30. Ludewig U, Neuhäuser B, Dynowsky M (2007) Molecular mechanisms of ammonium transport and accumulation in plants. FEBS Lett 581:2301–2308PubMedCrossRefGoogle Scholar
  31. Madigan M, Cox SS, Stegeman RA (1984) Nitrogen fixation and nitrogenase activities in members of the family Rhodospirillaceae. J Bacteriol 157:73–78PubMedGoogle Scholar
  32. Marini AM, Soussi-Boudekou S, Vissers S, André B (1997) A family of ammonium transporters in Saccharomyces cerevisiae. Mol Cell Biol 17:4282–4293PubMedGoogle Scholar
  33. Marini AM, Boeckstaens M, André B (2006) From yeast ammonium transporters to Rhesus proteins, isolation and functional characterization. Transfus Clin Biol 13:95–96PubMedCrossRefGoogle Scholar
  34. Martinez-Argudo I, Little R, Shearer N, Johnson P, Dixon R (2004) The NifL–NifA system: A multidomain transcriptional regulatory complex that integrates environmental signals. J Bacteriol 184:601–610CrossRefGoogle Scholar
  35. Masepohl B, Forchhammer K (2007) Regulatory cascades to express nitrogenase. In: Bothe H, Ferguson SJ, Newton WE (eds) Biology of the nitrogen cycle. Elsevier, Amsterdam pp. 131–145CrossRefGoogle Scholar
  36. Masepohl B, Kaiser B, Isakovic N, Richard CL, Kranz RG, Klipp W (2001) Urea utilization in the phototrophic bacterium Rhodobacter capsulatus is regulated by the transcriptional activator NtrC. J Bacteriol 183:637–643PubMedCrossRefGoogle Scholar
  37. Masepohl B, Klipp W (1996) Organization and regulation of genes encoding the molybdenum nitrogenase and the alternative nitrogenase in Rhodobacter capsulatus. Arch Microbiol 165: 80–90CrossRefGoogle Scholar
  38. Masepohl B, Klipp W, Pühler A (1988) Genetic characterization and sequence analysis of the duplicated nifA/nifB gene region of Rhodobacter capsulatus. Mol Gen Genet 212:27–37PubMedCrossRefGoogle Scholar
  39. Masepohl B, Kranz RG (2009) Regulation of nitrogen fixation. In: Hunter CN, Daldal F, Thurnauer MC, Beatty JT (eds) The purple phototrophic bacteria. Springer, Dordrecht pp. 759–775CrossRefGoogle Scholar
  40. Masepohl B, Krey R, Klipp W (1993) The draTG gene region of Rhodobacter capsulatus is required for post-translational regulation of both the molybdenum and the alternative nitrogenase. J Gen Microbiol 139:2667–2675PubMedCrossRefGoogle Scholar
  41. Masepohl B, Schneider K, Drepper T, Müller A, Klipp W (2002) Alternative nitrogenases. In: Leigh GJ (ed) Nitrogen fixation at the millennium. Elsevier, Amsterdam pp. 191–222CrossRefGoogle Scholar
  42. Maynard RH, Premakumar R, Bishop PE (1994) Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum. J Bacteriol 176:5583–5586PubMedGoogle Scholar
  43. Oda Y, Samanta SK, Rey FE, Wu L, Liu X, Yan T, Zhou J, Harwood CS (2005) Functional genomic analysis of three nitrogenase isozymes in the photosynthetic bacterium Rhodopseudomonas palustris. J Bacteriol 187:7784–7794PubMedCrossRefGoogle Scholar
  44. Paschen A, Drepper T, Masepohl B, Klipp W (2001) Rhodobacter capsulatus nifA mutants mediating nif gene expression in the presence of ammonium. FEMS Microbiol Lett 200:207–213PubMedCrossRefGoogle Scholar
  45. Pau RN (2004) Molybdenum uptake and homeostasis. In: Klipp W, Masepohl B, Gallon JR, Newton WE (eds) Genetics and regulation of nitrogen fixation in free-living bacteria. Kluwer, Dordrecht pp. 225–256Google Scholar
  46. Pawlowski A, Riedel K-U, Klipp W, Dreiskemper P, Groß S, Bierhoff H, Drepper T, Masepohl B (2003) Yeast two-hybrid studies on interaction of proteins involved in regulation of nitrogen fixation in the phototrophic bacterium Rhodobacter capsulatus. J Bacteriol 185: 5240–5247PubMedCrossRefGoogle Scholar
  47. Pierrard JP, Ludden PW, Roberts GP (1993) Posttranslational regulation of nitrogenase in Rhodobacter capsulatus: Existence of two independent regulatory effects of ammonium. J Bacteriol 175:1358–1366PubMedGoogle Scholar
  48. Pioszak AA, Ninfa AJ (2004) Mutations altering the N-terminal receiver domain of NRI (NtrC) that prevent dephosphorylation by the NRII-PII complex in Escherichia coli. J Bacteriol 186: 5730–5740PubMedCrossRefGoogle Scholar
  49. Preker P, Hübner P, Schmehl M, Klipp W, Bickle TA (1992) Mapping and characterization of the promoter elements of the regulatory nif genes rpoN, nifA1 and nifA2 in Rhodobacter capsulatus. Mol Microbiol 6:1035–1047PubMedCrossRefGoogle Scholar
  50. Rey FE, Heiniger EK, Harwood CS (2007) Redirection of metabolism for biological hydrogen production. Appl Environ Microbiol 73:1665–1671PubMedCrossRefGoogle Scholar
  51. Rutherford JC, Chua G, Hughes T, Cardenas ME, Heitman J (2008) A Mep2-dependent transcriptional profile links permease function to gene expression during pseudohyphal growth in Saccharomyces cerevisiae. Mol Biol Cell 19:3028–3039PubMedCrossRefGoogle Scholar
  52. Schmehl M, Jahn A, Meyer zu Vilsendorf A, Hennecke S, Masepohl B, Schuppler M, Marxer M, Oelze J, Klipp W (1993) Identification of a new class of nitrogen fixation genes in Rhodobacter capsulatus: A putative membrane complex involved in electron transport to nitrogenase. Mol Gen Genet 241:602–615PubMedCrossRefGoogle Scholar
  53. Schneider K, Gollan U, Selsemeier-Voigt S, Plass W, Müller A (1994) Rapid purification of the protein components of a highly active “iron only” nitrogenase. Naturwissenschaften 81: 405–408PubMedCrossRefGoogle Scholar
  54. Schüddekopf K, Hennecke S, Liese U, Kutsche M, Klipp W (1993) Characterization of anf genes specific for the alternative nitrogenase and identification of nif genes required for both nitrogenases in Rhodobacter capsulatus. Mol Microbiol 8:673–684PubMedCrossRefGoogle Scholar
  55. Schüttelkopf AW, Boxer DH, Hunter WN (2003) Crystal structure of activated ModE reveals conformational changes involving both oxyanion and DNA-binding domains. J Mol Biol 326:761–767PubMedCrossRefGoogle Scholar
  56. Seefeldt LC, Hoffman BM, Dean DR (2009) Mechanism of Mo-dependent nitrogenase. Annu Rev Biochem 78:701–722PubMedCrossRefGoogle Scholar
  57. Self WT, Grunden AM, Hasona A, Shanmugam KT (2001) Molybdate transport. Res Microbiol 152:311–321PubMedCrossRefGoogle Scholar
  58. Soupene E, He L, Yan D, Kustu S (1998) Ammonia acquisition in enteric bacteria: physiological role of the ammonium/methyl ammonium transport B (AmtB) protein. Proc Natl Acad Sci USA 95:7030–7034PubMedCrossRefGoogle Scholar
  59. Tremblay P-L, Drepper T, Masepohl B, Hallenbeck PC (2007) Membrane sequestration of PII proteins and nitrogenase regulation in the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol 189:5850–5859PubMedCrossRefGoogle Scholar
  60. Tremblay P-L, Hallenbeck PC (2008) Ammonia-induced formation of an AmtB-GlnK complex is not sufficient for nitrogenase regulation in the photosynthetic bacterium Rhodobacter capsulatus. J Bacteriol 190:1588–1594PubMedCrossRefGoogle Scholar
  61. Tremblay P-L, Hallenbeck PC (2009) Of blood, brains and bacteria, the Amt/Rh transporter family: Emerging role of Amt as a unique microbial sensor. Mol Microbiol 71:12–22PubMedCrossRefGoogle Scholar
  62. Wang G, Angermüller S, Klipp W (1993) Characterization of Rhodobacter capsulatus genes encoding a molybdenum transport system and putative molybdenum–pterin-binding proteins. J Bacteriol 175:3031–3042PubMedGoogle Scholar
  63. Wang H, Franke CC, Nordlund S, Norén A (2005) Reversible membrane association of dinitrogenase reductase activating glycohydrolase in the regulation of nitrogenase activity in Rhodospirillum rubrum; dependence on GlnJ and AmtB1. FEMS Microbiol Lett 253: 273–279PubMedCrossRefGoogle Scholar
  64. Wiethaus J, Müller A, Neumann M, Neumann S, Leimkühler S, Narberhaus F, Masepohl B (2009) Specific interactions between four molybdenum-binding proteins contribute to Mo-dependent gene regulation in Rhodobacter capsulatus. J Bacteriol 191:5205–5215PubMedCrossRefGoogle Scholar
  65. Wiethaus J, Wirsing A, Narberhaus F, Masepohl B (2006) Overlapping and specialized functions of the molybdenum-dependent regulators MopA and MopB in Rhodobacter capsulatus. J Bacteriol 188:8441–8451PubMedCrossRefGoogle Scholar
  66. Wolfe DM, Zhang Y, Roberts GP (2007) Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum. J Bacteriol 189:6861–6869PubMedCrossRefGoogle Scholar
  67. Yakunin AF, Fedorov AS, Laurinavichene TV, Glaser VM, Egorov NS, Tsygankov A, Zinchenko VS, Hallenbeck PC (2001) Regulation of nitrogenase in the photosynthetic bacterium Rhodobacter sphaeroides containing draTG and nifHDK genes from Rhodobacter capsulatus. Can J Microbiol 47:206–212PubMedGoogle Scholar
  68. Yakunin AF, Hallenbeck PC (1998) Short-term nitrogenase regulation in Rhodobacter capsulatus: Multiple in vivo nitrogenase responses to NH4 + addition. J Bacteriol 180:6392–6395PubMedGoogle Scholar
  69. Yakunin AF, Hallenbeck PC (1999) The presence of ADP-ribosylated Fe protein of nitrogenase in Rhodobacter capsulatus is correlated with the cellular nitrogen status. J Bacteriol 181: 1994–2000PubMedGoogle Scholar
  70. Yakunin AF, Hallenbeck PC (2000) Regulation of nitrogenase activity in Rhodobacter capsulatus under dark microoxic conditions. Arch Microbiol 173:366–372PubMedCrossRefGoogle Scholar
  71. Yakunin AF, Hallenbeck PC (2002) AmtB is necessary for NH4 +-induced nitrogenase switch-off and ADP-ribosylation in Rhodobacter capsulatus. J Bacteriol 184:4081–4088PubMedCrossRefGoogle Scholar
  72. Yildiz O, Kalthoff C, Raunser, Kuhlbrandt W (2007) Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake. EMBO J 26:589–599PubMedCrossRefGoogle Scholar
  73. Zhang Y, Cummings AD, Burris RH, Ludden PW, Roberts GP (1995) Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum. J Bacteriol 177:5322–5326PubMedGoogle Scholar
  74. Zhang Y, Pohlmann EL, Ludden PW, Roberts GP (2000) Mutagenesis and functional characterization of the glnB, glnA, and nifA genes from the photosynthetic bacterium Rhodospirillum rubrum. J Bacteriol 182:983–992PubMedCrossRefGoogle Scholar
  75. Zhang Y, Pohlmann EL, Ludden PW, Roberts GP (2001) Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: Roles in sensing ammonium and energy status. J Bacteriol 183:6159–6168PubMedCrossRefGoogle Scholar
  76. Zhang Y, Pohlmann EL, Roberts GP (2005) GlnD is essential for NifA activation, NtrB/NtrC-regulated gene expression, and posttranslational regulation of nitrogenase activity in the photosynthetic, nitrogen-fixing bacterium Rhodospirillum rubrum. J Bacteriol 187:1254–1265PubMedCrossRefGoogle Scholar
  77. Zhang Y, Wolfe DM, Pohlmann EL, Conrad MC, Roberts GP (2006) Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum. Microbiology 152:2075–2089PubMedCrossRefGoogle Scholar
  78. Zhu Y, Conrad MC, Zhang Y, Roberts GP (2006) Identification of Rhodospirillum rubrum GlnB variants that are altered in their ability to interact with different targets in response to nitrogen status signals. J Bacteriol 188:1866–1874PubMedCrossRefGoogle Scholar
  79. Zinchenko V, Babykin M, Glaser S, Mekhedov S, Shestakov S (1997) Mutation in ntrC gene leading to the derepression of nitrogenase synthesis in Rhodobacter sphaeroides. FEMS Microbiol Lett 147:57–61PubMedCrossRefGoogle Scholar
  80. Zou X, Zhu Y, Pohlmann EL, Li J, Zhang Y, Roberts GP (2008) Identification and functional characterization of NifA variants that are independent of GlnB activation in the photosynthetic bacterium Rhodospirillum rubrum. Microbiology 154:2689–2699PubMedCrossRefGoogle Scholar

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Authors and Affiliations

  1. 1.Lehrstuhl für Biologie der Mikroorganismen, Fakultät für Biologie und BiotechnologieRuhr-Universität BochumBochumGermany
  2. 2.Département de Microbiologie et ImmunologieUniversité de MontréalMontréalCanada

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