1887

Abstract

The PII family of signal transduction proteins is widespread amongst the three domains of life, and its members have fundamental roles in the general control of nitrogen metabolism. These proteins exert their regulatory role by direct protein–protein interaction with a multitude of cellular targets. The interactions are dependent on the binding of metabolites such as ATP, ADP and 2-oxoglutarate (2-OG), and on whether or not the PII protein is modified. In the photosynthetic nitrogen-fixing bacterium three PII paralogues have been identified and termed GlnB, GlnJ and GlnK. In this report we analysed the interaction of GlnJ with known cellular targets such as the ammonium transporter AmtB1, the adenylyltransferase GlnE and the uridylyltransferase GlnD. Our results show that the interaction of GlnJ with cellular targets is regulated by the concentrations of manganese and 2-OG and the ADP : ATP ratio. Furthermore, we show here for the first time, to our knowledge, that in the interactions of GlnJ with the three different partners, the energy signal (ADP : ATP ratio) in fact overrides the carbon/nitrogen signal (2-OG). In addition, by generating specific amino acid substitutions in GlnJ we show that the interactions with different cellular targets are differentially affected, and the possible implications of these results are discussed. Our results are important to further the understanding of the regulatory role of PII proteins in , a photosynthetic bacterium in which the nitrogen fixation process and its intricate control mechanisms make the regulation of nitrogen metabolism even more complex than in other studied bacteria.

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2008-08-01
2019-10-14
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References

  1. Arcondeguy, T., Jack, R. & Merrick, M. ( 2001; ). P(II) signal transduction proteins, pivotal players in microbial nitrogen control. Microbiol Mol Biol Rev 65, 80–105.[CrossRef]
    [Google Scholar]
  2. Atkinson, M. R. & Ninfa, A. J. ( 1999; ). Characterization of the GlnK protein of Escherichia coli. Mol Microbiol 32, 301–313.[CrossRef]
    [Google Scholar]
  3. Bueno, R., Pahel, G. & Magasanik, B. ( 1985; ). Role of glnB and glnD gene products in regulation of the glnALG operon of Escherichia coli. J Bacteriol 164, 816–822.
    [Google Scholar]
  4. Conroy, M. J., Durand, A., Lupo, D., Li, X. D., Bullough, P. A., Winkler, F. K. & Merrick, M. ( 2007; ). The crystal structure of the Escherichia coli AmtB–GlnK complex reveals how GlnK regulates the ammonia channel. Proc Natl Acad Sci U S A 104, 1213–1218.[CrossRef]
    [Google Scholar]
  5. Durand, A. & Merrick, M. ( 2006; ). In vitro analysis of the Escherichia coli AmtB–GlnK complex reveals a stoichiometric interaction and sensitivity to ATP and 2-oxoglutarate. J Biol Chem 281, 29558–29567.[CrossRef]
    [Google Scholar]
  6. Forchhammer, K. & Tandeau de Marsac, N. ( 1994; ). The PII protein in the cyanobacterium Synechococcus sp. strain PCC 7942 is modified by serine phosphorylation and signals the cellular N-status. J Bacteriol 176, 84–91.
    [Google Scholar]
  7. Forchhammer, K., Irmler, A., Kloft, N. & Ruppert, U. ( 2004; ). PII signalling in unicellular cyanobacteria: analysis of redox-signals and energy charge. Physiol Plant 120, 51–56.[CrossRef]
    [Google Scholar]
  8. Gruswitz, F., O'Connell, J., III & Stroud, R. M. ( 2007; ). Inhibitory complex of the transmembrane ammonia channel, AmtB, and the cytosolic regulatory protein, GlnK, at 1.96 Å. Proc Natl Acad Sci U S A 104, 42–47.[CrossRef]
    [Google Scholar]
  9. Heinrich, A., Woyda, K., Brauburger, K., Meiss, G., Detsch, C., Stulke, J. & Forchhammer, K. ( 2006; ). Interaction of the membrane-bound GlnK–AmtB complex with the master regulator of nitrogen metabolism TnrA in Bacillus subtilis. J Biol Chem 281, 34909–34917.[CrossRef]
    [Google Scholar]
  10. Hesketh, A., Fink, D., Gust, B., Rexer, H. U., Scheel, B., Chater, K., Wohlleben, W. & Engels, A. ( 2002; ). The GlnD and GlnK homologues of Streptomyces coelicolor A3(2) are functionally dissimilar to their nitrogen regulatory system counterparts from enteric bacteria. Mol Microbiol 46, 319–330.[CrossRef]
    [Google Scholar]
  11. Huergo, L. F., Souza, E. M., Araujo, M. S., Pedrosa, F. O., Chubatsu, L. S., Steffens, M. B. & Merrick, M. ( 2006; ). ADP-ribosylation of dinitrogenase reductase in Azospirillum brasilense is regulated by AmtB-dependent membrane sequestration of DraG. Mol Microbiol 59, 326–337.[CrossRef]
    [Google Scholar]
  12. Huergo, L. F., Merrick, M., Pedrosa, F. O., Chubatsu, L. S., Araujo, L. M. & Souza, E. M. ( 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, 1523–1535.
    [Google Scholar]
  13. Javelle, A., Severi, E., Thornton, J. & Merrick, M. ( 2004; ). Ammonium sensing in Escherichia coli. Role of the ammonium transporter AmtB and AmtB–GlnK complex formation. J Biol Chem 279, 8530–8538.[CrossRef]
    [Google Scholar]
  14. Jiang, P. & Ninfa, A. J. ( 2007; ). Escherichia coli PII signal transduction protein controlling nitrogen assimilation acts as a sensor of adenylate energy charge in vitro. Biochemistry 46, 12979–12996.[CrossRef]
    [Google Scholar]
  15. Jiang, P., Zucker, P., Atkinson, M. R., Kamberov, E. S., Tirasophon, W., Chandran, P., Schefke, B. R. & Ninfa, A. J. ( 1997; ). Structure/function analysis of the PII signal transduction protein of Escherichia coli: genetic separation of interactions with protein receptors. J Bacteriol 179, 4342–4353.
    [Google Scholar]
  16. Jiang, P., Peliska, J. A. & Ninfa, A. J. ( 1998a; ). The regulation of Escherichia coli glutamine synthetase revisited: role of 2-ketoglutarate in the regulation of glutamine synthetase adenylylation state. Biochemistry 37, 12802–12810.[CrossRef]
    [Google Scholar]
  17. Jiang, P., Peliska, J. A. & Ninfa, A. J. ( 1998b; ). Enzymological characterization of the signal-transducing uridylyltransferase/uridylyl-removing enzyme (EC 2.7.7.59) of Escherichia coli and its interaction with the PII protein. Biochemistry 37, 12782–12794.[CrossRef]
    [Google Scholar]
  18. Johansson, M. & Nordlund, S. ( 1996; ). Transcription of the glnB and glnA genes in the photosynthetic bacterium Rhodospirillum rubrum. Microbiology 142, 1265–1272.[CrossRef]
    [Google Scholar]
  19. Johansson, M. & Nordlund, S. ( 1999; ). Purification of P(II) and P(II)–UMP and in vitro studies of regulation of glutamine synthetase in Rhodospirillum rubrum. J Bacteriol 181, 6524–6529.
    [Google Scholar]
  20. Jonsson, A. & Nordlund, S. ( 2007; ). In vitro studies of the uridylylation of the three PII protein paralogs from Rhodospirillum rubrum: the transferase activity of R. rubrum GlnD is regulated by α-ketoglutarate and divalent cations but not by glutamine. J Bacteriol 189, 3471–3478.[CrossRef]
    [Google Scholar]
  21. Jonsson, A., Teixeira, P. F. & Nordlund, S. ( 2007; ). The activity of adenylyltransferase in Rhodospirillum rubrum is only affected by α-ketoglutarate and unmodified PII proteins, but not by glutamine, in vitro. FEBS J 274, 2449–2460.[CrossRef]
    [Google Scholar]
  22. Kamberov, E. S., Atkinson, M. R. & Ninfa, A. J. ( 1995; ). The Escherichia coli PII signal transduction protein is activated upon binding 2-ketoglutarate and ATP. J Biol Chem 270, 17797–17807.[CrossRef]
    [Google Scholar]
  23. Khademi, S., O'Connell, J., III, Remis, J., Robles-Colmenares, Y., Miercke, L. J. & Stroud, R. M. ( 2004; ). Mechanism of ammonia transport by Amt/MEP/Rh: structure of AmtB at 1.35 Å. Science 305, 1587–1594.[CrossRef]
    [Google Scholar]
  24. Laemmli, U. K. ( 1970; ). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef]
    [Google Scholar]
  25. Li, J. D., Hu, C. Z. & Yoch, D. C. ( 1987; ). Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by or darkness. J Bacteriol 169, 231–237.
    [Google Scholar]
  26. Lowry, O. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. ( 1951; ). Protein measurement with the Folin phenol reagent. J Biol Chem 193, 265–275.
    [Google Scholar]
  27. Maheswaran, M., Urbanke, C. & Forchhammer, K. ( 2004; ). Complex formation and catalytic activation by the PII signaling protein of N-acetyl-l-glutamate kinase from Synechococcus elongatus strain PCC 7942. J Biol Chem 279, 55202–55210.[CrossRef]
    [Google Scholar]
  28. Merrick, M. J. & Edwards, R. A. ( 1995; ). Nitrogen control in bacteria. Microbiol Rev 59, 604–622.
    [Google Scholar]
  29. Nordlund, S. & Höglund, L. ( 1986; ). Studies of the adenylate and pyridine nucleotide pools during nitrogenase “switch-off” in Rhodospirillum rubrum. Plant Soil 90, 203–209.[CrossRef]
    [Google Scholar]
  30. Nordlund, S. & Ludden, P. W. ( 2004; ). Post-translational regulation of nitrogenase in photosynthetic bacteria. In Genetics and Regulation of Nitrogen Fixation in Free-Living Bacteria, pp. 175–196. Edited by W. Klipp, B. Masephol, J. R. Gallon & W. E. Newton. Dordrecht, The Netherlands: Kluwer Academic Publishers.
  31. Ormerod, J. G., Ormerod, K. S. & 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, 449–463.[CrossRef]
    [Google Scholar]
  32. Paul, T. D. & Ludden, P. W. ( 1984; ). Adenine nucleotide levels in Rhodospirillum rubrum during switch-off of whole-cell nitrogenase activity. Biochem J 224, 961–969.
    [Google Scholar]
  33. Pioszak, A. A., Jiang, P. & Ninfa, A. J. ( 2000; ). The Escherichia coli PII signal transduction protein regulates the activities of the two-component system transmitter protein NRII by direct interaction with the kinase domain of the transmitter module. Biochemistry 39, 13450–13461.[CrossRef]
    [Google Scholar]
  34. Smith, C. S., Morrice, N. A. & Moorhead, G. B. ( 2004; ). Lack of evidence for phosphorylation of Arabidopsis thaliana PII: implications for plastid carbon and nitrogen signaling. Biochim Biophys Acta 1699, 145–154.[CrossRef]
    [Google Scholar]
  35. Strosser, J., Ludke, A., Schaffer, S., Kramer, R. & Burkovski, A. ( 2004; ). Regulation of GlnK activity: modification, membrane sequestration and proteolysis as regulatory principles in the network of nitrogen control in Corynebacterium glutamicum. Mol Microbiol 54, 132–147.[CrossRef]
    [Google Scholar]
  36. Thomas, G., Coutts, G. & Merrick, M. ( 2000; ). The glnKamtB operon. A conserved gene pair in prokaryotes. Trends Genet 16, 11–14.
    [Google Scholar]
  37. Wang, H., Franke, C. C., Nordlund, S. & Noren, 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, 273–279.[CrossRef]
    [Google Scholar]
  38. Wolfe, D. M., Zhang, Y. & Roberts, G. P. ( 2007; ). Specificity and regulation of interaction between the PII and AmtB1 proteins in Rhodospirillum rubrum. J Bacteriol 189, 6861–6869.[CrossRef]
    [Google Scholar]
  39. Xu, Y., Cheah, E., Carr, P. D., van Heeswijk, W. C., Westerhoff, H. V., Vasudevan, S. G. & Ollis, D. L. ( 1998; ). GlnK, a PII-homologue: structure reveals ATP binding site and indicates how the T-loops may be involved in molecular recognition. J Mol Biol 282, 149–165.[CrossRef]
    [Google Scholar]
  40. Yildiz, O., Kalthoff, C., Raunser, S. & Kuhlbrandt, W. ( 2007; ). Structure of GlnK1 with bound effectors indicates regulatory mechanism for ammonia uptake. EMBO J 26, 589–599.[CrossRef]
    [Google Scholar]
  41. Zhang, Y., Pohlmann, E. L., Ludden, P. W. & Roberts, G. P. ( 2001; ). Functional characterization of three GlnB homologs in the photosynthetic bacterium Rhodospirillum rubrum: roles in sensing ammonium and energy status. J Bacteriol 183, 6159–6168.[CrossRef]
    [Google Scholar]
  42. Zhang, Y., Pohlmann, E. L. & Roberts, G. P. ( 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, 1254–1265.[CrossRef]
    [Google Scholar]
  43. Zhang, Y., Pohlmann, E. L., Conrad, M. C. & Roberts, G. P. ( 2006a; ). The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity. Mol Microbiol 61, 497–510.[CrossRef]
    [Google Scholar]
  44. Zhang, Y., Wolfe, D. M., Pohlmann, E. L., Conrad, M. C. & Roberts, G. P. ( 2006b; ). Effect of AmtB homologues on the post-translational regulation of nitrogenase activity in response to ammonium and energy signals in Rhodospirillum rubrum. Microbiology 152, 2075–2089.[CrossRef]
    [Google Scholar]
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