The nitrogen interaction network in WH5701, a cyanobacterium with two PipX and two P-like proteins Free

Abstract

Nitrogen regulation involves the formation of different types of protein complexes between signal transducers and their transcriptional or metabolic targets. In oxygenic phototrophs, the signal integrator P activates the enzyme -acetyl--glutamate kinase (NAGK) by complex formation. P also interacts with PipX, a protein with a tudor-like domain that mediates contacts with P and with the transcriptional regulator NtcA, to which it binds to increase its activity. Here, we use a combination of , yeast two-hybrid and approaches to investigate the nitrogen regulation network of WH5701, a marine cyanobacterium with two P (GlnB_A and GlnB_B) and two PipX (PipX_I and PipX_II) proteins. Our results indicate that GlnB_A is functionally equivalent to the canonical P protein from . GlnB_A interacted with PipX and NAGK proteins and stimulated NAGK activity, counteracting arginine inhibition. GlnB_B had only a slight stimulatory effect on NAGK activity, but its potential to bind effectors and form heterotrimers in WH5701 indicates additional regulatory functions. PipX_II, and less evidently PipX_I, specifically interacted with GlnB_A and NtcA, supporting a role for both WH5701 PipX proteins in partner swapping with GlnB_A and NtcA.

Loading

Article metrics loading...

/content/journal/micro/10.1099/mic.0.047266-0
2011-04-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/157/4/1220.html?itemId=/content/journal/micro/10.1099/mic.0.047266-0&mimeType=html&fmt=ahah

References

  1. Ausubel F. M., Brent R., Kingston R. E., Moore D. editors 1999 Short Protocols in Molecular Biology: a Compendium of Methods from Current Protocols in Molecular Biology New York: Wiley;
    [Google Scholar]
  2. Bartel P., Chien C. T., Sternglanz R., Fields S. 1993; Using the two hybrid system to detect protein-protein interactions. In Cellular Interactions in Development: A Practical Approach pp 153–179 Edited by Hartley D. A. Oxford, UK: Oxford University Press;
    [Google Scholar]
  3. Beez S., Fokina O., Herrmann C., Forchhammer K. 2009; N -acetyl-l-glutamate kinase (NAGK) from oxygenic phototrophs: PII signal transduction across domains of life reveals novel insights in NAGK control. J Mol Biol 389:748–758
    [Google Scholar]
  4. Burillo S., Luque I., Fuentes I., Contreras A. 2004; Interactions between the nitrogen signal transduction protein PII and N -acetyl glutamate kinase in organisms that perform oxygenic photosynthesis. J Bacteriol 186:3346–3354
    [Google Scholar]
  5. Chen Y. M., Ferrar T. S., Lohmeier-Vogel E. M., Morrice N., Mizuno Y., Berenger B., Ng K. K., Muench D. G., Moorhead G. B. 2006; The PII signal transduction protein of Arabidopsis thaliana forms an arginine-regulated complex with plastid N -acetyl glutamate kinase. J Biol Chem 281:5726–5733
    [Google Scholar]
  6. Espinosa J., Forchhammer K., Burillo S., Contreras A. 2006; Interaction network in cyanobacterial nitrogen regulation: PipX, a protein that interacts in a 2-oxoglutarate dependent manner with PII and NtcA. Mol Microbiol 61:457–469
    [Google Scholar]
  7. Espinosa J., Forchhammer K., Contreras A. 2007; Role of the Synechococcus PCC 7942 nitrogen regulator protein PipX in NtcA-controlled processes. Microbiology 153:711–718
    [Google Scholar]
  8. Espinosa J., Castells M. A., Laichoubi K. B., Contreras A. 2009; Mutations at pipX suppress lethality of PII-deficient mutants of Synechococcus elongatus PCC 7942. J Bacteriol 191:4863–4869
    [Google Scholar]
  9. Espinosa J., Castells M. A., Laichoubi K. B., Forchhammer K., Contreras A. 2010; Effects of spontaneous mutations in PipX functions and regulatory complexes on the cyanobacterium Synechococcus elongatus strain PCC 7942. Microbiology 156:1517–1526
    [Google Scholar]
  10. Fokina O., Chellamuthu V. R., Forchhammer K., Zeth K. 2010a; Mechanism of 2-oxoglutarate signaling by the Synechococcus elongatus PII signal transduction protein. Proc Natl Acad Sci U S A 107:19760–19765
    [Google Scholar]
  11. Fokina O., Chellamuthu V. R., Zeth K., Forchhammer K. 2010b; A novel signal transduction protein PII variant from Synechococcus elongatus PCC 7942 indicates a two-step process for NAGK-PII complex formation. J Mol Biol 399:410–421
    [Google Scholar]
  12. Forchhammer K. 2004; Global carbon/nitrogen control by PII signal transduction in cyanobacteria: from signals to targets. FEMS Microbiol Rev 28:319–333
    [Google Scholar]
  13. Forchhammer K. 2008; PII signal transducers: novel functional and structural insights. Trends Microbiol 16:65–72
    [Google Scholar]
  14. Forchhammer K., Hedler A., Strobel H., Weiss V. 1999; Heterotrimerization of PII-like signalling proteins: implications for PII-mediated signal transduction systems. Mol Microbiol 33:338–349
    [Google Scholar]
  15. Hanahan D. 1985; Techniques for transformation of Escherichia coli . In DNA Cloning pp 109–135 Edited by Glover D. Oxford, UK: IRL Press;
    [Google Scholar]
  16. Harper J. W., Adami G. R., Wei N., Keyomarsi K., Elledge S. J. 1993; The p21 Cdk-interacting protein Cip1 is a potent inhibitor of G1 cyclin-dependent kinases. Cell 75:805–816
    [Google Scholar]
  17. Heinrich A., Maheswaran M., Ruppert U., Forchhammer K. 2004; The Synechococcus elongatus PII signal transduction protein controls arginine synthesis by complex formation with N -acetyl-l-glutamate kinase. Mol Microbiol 52:1303–1314
    [Google Scholar]
  18. Herrero A., Muro-Pastor A. M., Flores E. 2001; Nitrogen control in cyanobacteria. J Bacteriol 183:411–425
    [Google Scholar]
  19. James P., Halladay J., Craig E. A. 1996; Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144:1425–1436
    [Google Scholar]
  20. Larkin M. A., Blackshields G., Brown N. P., Chenna R., McGettigan P. A., McWilliam H., Valentin F., Wallace I. M., Wilm A. other authors 2007; clustal w and clustal_x version 2.0. Bioinformatics 23:2947–2948
    [Google Scholar]
  21. Laurent S., Chen H., Bédu S., Ziarelli F., Peng L., Zhang C. C. 2005; Nonmetabolizable analogue of 2-oxoglutarate elicits heterocyst differentiation under repressive conditions in Anabaena sp. PCC 7120. Proc Natl Acad Sci U S A 1029907–9912
    [Google Scholar]
  22. Leigh J. A., Dodsworth J. A. 2007; Nitrogen regulation in bacteria and archaea. Annu Rev Microbiol 61:349–377
    [Google Scholar]
  23. Llácer J. L., Contreras A., Forchhammer K., Marco-Marín C., Gil-Ortiz F., Maldonado R., Fita I., Rubio V. 2007; The crystal structure of the complex of PII and acetylglutamate kinase reveals how PII controls the storage of nitrogen as arginine. Proc Natl Acad Sci U S A 104:17644–17649
    [Google Scholar]
  24. Llácer J. L., Espinosa J., Castells M. A., Contreras A., Forchhammer K., Rubio V. 2010; Structural basis for the regulation of NtcA-dependent transcription by proteins PipX and PII. Proc Natl Acad Sci U S A 107:15397–15402
    [Google Scholar]
  25. Luque I., Zabulon G., Contreras A., Houmard J. 2001; Convergence of two global transcriptional regulators on nitrogen induction of the stress-acclimation gene nblA in the cyanobacterium Synechococcus sp. PCC 7942. Mol Microbiol 41:937–947
    [Google Scholar]
  26. 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
    [Google Scholar]
  27. Muro-Pastor M. I., Reyes J. C., Florencio F. J. 2001; Cyanobacteria perceive nitrogen status by sensing intracellular 2-oxoglutarate levels. J Biol Chem 276:38320–38328
    [Google Scholar]
  28. Muro-Pastor M. I., Reyes J. C., Florencio F. J. 2005; Ammonium assimilation in cyanobacteria. Photosynth Res 83:135–150
    [Google Scholar]
  29. Palinska K. A., Laloui W., Bédu, S., Loiseaux-de Goër S., Castets A. M., Rippka R., Tandeau de Marsac N. 2002; The signal transducer PII and bicarbonate acquisition in Prochlorococcus marinus PCC 9511, a marine cyanobacterium naturally deficient in nitrate and nitrite assimilation. Microbiology 148:2405–2412
    [Google Scholar]
  30. Phillips T. A., VanBogelen R. A., Neidhardt F. C. 1984; lon gene product of Escherichia coli is a heat-shock protein. J Bacteriol 159:283–287
    [Google Scholar]
  31. Sant'Anna F. H., Trentini D. B., de Souto Weber S., Cecagno R., da Silva S. C., Schrank I. S. 2009; The PII superfamily revised: a novel group and evolutionary insights. J Mol Evol 68:322–336
    [Google Scholar]
  32. Sauer J., Dirmeier U., Forchhammer K. 2000; The Synechococcus strain PCC 7942 glnN product (glutamine synthetase III) helps recovery from prolonged nitrogen chlorosis. J Bacteriol 182:5615–5619
    [Google Scholar]
  33. Scanlan D. J., Ostrowski M., Mazard S., Dufresne A., Garczarek L., Hess W. R., Post A. F., Hagemann M., Paulsen I., Partensky F. 2009; Ecological genomics of marine picocyanobacteria. Microbiol Mol Biol Rev 73:249–299
    [Google Scholar]
  34. Sugiyama K., Hayakawa T., Kudo T., Ito T., Yamaya T. 2004; Interaction of N-acetylglutamate kinase with a PII-like protein in rice. Plant Cell Physiol 45:1768–1778
    [Google Scholar]
  35. Tanigawa R., Shirokane M., Maeda Si S., Omata T., Tanaka K., Takahashi H. 2002; Transcriptional activation of NtcA-dependent promoters of Synechococcus sp. PCC 7942 by 2-oxoglutarate in vitro . Proc Natl Acad Sci U S A 99:4251–4255
    [Google Scholar]
  36. Vázquez-Bermúdez M. F., Herrero A., Flores E. 2002; 2-Oxoglutarate increases the binding affinity of the NtcA (nitrogen control) transcription factor for the Synechococcus glnA promoter. FEBS Lett 512:71–74
    [Google Scholar]
  37. Zhao M. X., Jiang Y. L., Xu B. Y., Chen Y., Zhang C. C., Zhou C. Z. 2010; Crystal structure of the cyanobacterial signal transduction protein PII in complex with PipX. J Mol Biol 402:552–559
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/mic.0.047266-0
Loading
/content/journal/micro/10.1099/mic.0.047266-0
Loading

Data & Media loading...

Supplements

Supplementary material 1

PDF

Most cited Most Cited RSS feed