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. 1997 Apr;179(8):2678–2689. doi: 10.1128/jb.179.8.2678-2689.1997

Transcription of glutamine synthetase genes (glnA and glnN) from the cyanobacterium Synechocystis sp. strain PCC 6803 is differently regulated in response to nitrogen availability.

J C Reyes 1, M I Muro-Pastor 1, F J Florencio 1
PMCID: PMC179018  PMID: 9098067

Abstract

In the cyanobacterium Synechocystis sp. strain PCC 6803 we have previously reported the presence of two different proteins with glutamine synthetase activity: GSI, encoded by the glnA gene, and GSIII, encoded by the glnN gene. In this work we show that expression of both the glnA and glnN genes is subjected to transcriptional regulation in response to changes in nitrogen availability. Northern blot experiments and transcriptional fusions demonstrated that the glnA gene is highly transcribed in nitrate- or ammonium-grown cells and exhibits two- to fourfold-higher expression in nitrogen-starved cells. In contrast, the glnN gene is highly expressed only under nitrogen deficiency. Half-lives of both mRNAs, calculated after addition of rifampin or ammonium to nitrogen-starved cells, were not significantly different (2.5 or 3.4 min, respectively, for glnA mRNA; 1.9 or 1.4 min, respectively, for glnN mRNA), suggesting that changes in transcript stability are not involved in the regulation of the expression of both genes. Deletions of the glnA and glnN upstream regions were used to delimit the promoter and the regulatory sequences of both genes. Primer extension analysis showed that structure of the glnA gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified, Escherichia coli-expressed Synechocystis NtcA protein binds to the promoter of the glnA gene. Primer extension also revealed the existence of a sequence related to the NtcA binding site upstream from the glnN promoter. However, E. coli-expressed NtcA failed to bind to this site. These findings suggest that an additional modification of NtcA or an additional factor is required for the regulation of glnN gene expression.

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Selected References

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  1. Bovy A., de Vrieze G., Borrias M., Weisbeek P. Transcriptional regulation of the plastocyanin and cytochrome c553 genes from the cyanobacterium Anabaena species PCC 7937. Mol Microbiol. 1992 Jun;6(11):1507–1513. doi: 10.1111/j.1365-2958.1992.tb00871.x. [DOI] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Cai Y. P., Wolk C. P. Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120 to select for double recombinants and to entrap insertion sequences. J Bacteriol. 1990 Jun;172(6):3138–3145. doi: 10.1128/jb.172.6.3138-3145.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Carlson T. A., Martin G. B., Chelm B. K. Differential transcription of the two glutamine synthetase genes of Bradyrhizobium japonicum. J Bacteriol. 1987 Dec;169(12):5861–5866. doi: 10.1128/jb.169.12.5861-5866.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chiurazzi M., Iaccarino M. Transcriptional analysis of the glnB-glnA region of Rhizobium leguminosarum biovar viciae. Mol Microbiol. 1990 Oct;4(10):1727–1735. doi: 10.1111/j.1365-2958.1990.tb00550.x. [DOI] [PubMed] [Google Scholar]
  6. Chávez S., Candau P. An NAD-specific glutamate dehydrogenase from cyanobacteria. Identification and properties. FEBS Lett. 1991 Jul 8;285(1):35–38. doi: 10.1016/0014-5793(91)80719-j. [DOI] [PubMed] [Google Scholar]
  7. Cohen-Kupiec R., Gurevitz M., Zilberstein A. Expression of glnA in the cyanobacterium Synechococcus sp. strain PCC 7942 is initiated from a single nif-like promoter under various nitrogen conditions. J Bacteriol. 1993 Dec;175(23):7727–7731. doi: 10.1128/jb.175.23.7727-7731.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Elmorjani K., Liotenberg S., Houmard J., de Marsac N. T. Molecular characterization of the gene encoding glutamine synthetase in the cyanobacterium Calothrix sp. PCC 7601. Biochem Biophys Res Commun. 1992 Dec 30;189(3):1296–1302. doi: 10.1016/0006-291x(92)90214-6. [DOI] [PubMed] [Google Scholar]
  9. Ferino F., Chauvat F. A promoter-probe vector-host system for the cyanobacterium, Synechocystis PCC6803. Gene. 1989 Dec 14;84(2):257–266. doi: 10.1016/0378-1119(89)90499-x. [DOI] [PubMed] [Google Scholar]
  10. Frías J. E., Flores E., Herrero A. Requirement of the regulatory protein NtcA for the expression of nitrogen assimilation and heterocyst development genes in the cyanobacterium Anabaena sp. PCC 7120. Mol Microbiol. 1994 Nov;14(4):823–832. doi: 10.1111/j.1365-2958.1994.tb01318.x. [DOI] [PubMed] [Google Scholar]
  11. Frías J. E., Mérida A., Herrero A., Martín-Nieto J., Flores E. General distribution of the nitrogen control gene ntcA in cyanobacteria. J Bacteriol. 1993 Sep;175(17):5710–5713. doi: 10.1128/jb.175.17.5710-5713.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. García-Domínguez M., Reyes J. C., Florencio F. J. Purification and characterization of a new type of glutamine synthetase from cyanobacteria. Eur J Biochem. 1997 Feb 15;244(1):258–264. doi: 10.1111/j.1432-1033.1997.00258.x. [DOI] [PubMed] [Google Scholar]
  13. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  14. Hawley D. K., McClure W. R. Compilation and analysis of Escherichia coli promoter DNA sequences. Nucleic Acids Res. 1983 Apr 25;11(8):2237–2255. doi: 10.1093/nar/11.8.2237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ikeda T. P., Shauger A. E., Kustu S. Salmonella typhimurium apparently perceives external nitrogen limitation as internal glutamine limitation. J Mol Biol. 1996 Jun 21;259(4):589–607. doi: 10.1006/jmbi.1996.0342. [DOI] [PubMed] [Google Scholar]
  16. Leach C. K., Carr N. G. In vitro protein synthesis and measurement of the stability of messenger RNA in the blue-green alga, anabaena variabilis. J Gen Microbiol. 1974 Mar;81(1):47–58. doi: 10.1099/00221287-81-1-47. [DOI] [PubMed] [Google Scholar]
  17. Luque I., Flores E., Herrero A. Molecular mechanism for the operation of nitrogen control in cyanobacteria. EMBO J. 1994 Jun 15;13(12):2862–2869. doi: 10.1002/j.1460-2075.1994.tb06580.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Markwell M. A., Haas S. M., Bieber L. L., Tolbert N. E. A modification of the Lowry procedure to simplify protein determination in membrane and lipoprotein samples. Anal Biochem. 1978 Jun 15;87(1):206–210. doi: 10.1016/0003-2697(78)90586-9. [DOI] [PubMed] [Google Scholar]
  19. Meeks J. C., Wolk C. P., Lockau W., Schilling N., Shaffer P. W., Chien W. S. Pathways of assimilation of [13N]N2 and 13NH4+ by cyanobacteria with and without heterocysts. J Bacteriol. 1978 Apr;134(1):125–130. doi: 10.1128/jb.134.1.125-130.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Meeks J. C., Wolk C. P., Thomas J., Lockau W., Shaffer P. W., Austin S. M., Chien W. S., Galonsky A. The pathways of assimilation of 13NH4+ by the cyanobacterium, Anabaena cylindrica. J Biol Chem. 1977 Nov 10;252(21):7894–7900. [PubMed] [Google Scholar]
  21. Meissner P. S., Sisk W. P., Berman M. L. Bacteriophage lambda cloning system for the construction of directional cDNA libraries. Proc Natl Acad Sci U S A. 1987 Jun;84(12):4171–4175. doi: 10.1073/pnas.84.12.4171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Merrick M. J., Edwards R. A. Nitrogen control in bacteria. Microbiol Rev. 1995 Dec;59(4):604–622. doi: 10.1128/mr.59.4.604-622.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Mohamed A., Jansson C. Influence of light on accumulation of photosynthesis-specific transcripts in the cyanobacterium Synechocystis 6803. Plant Mol Biol. 1989 Dec;13(6):693–700. doi: 10.1007/BF00016024. [DOI] [PubMed] [Google Scholar]
  24. Mohamed A., Jansson C. Photosynthetic electron transport controls degradation but not production of psbA transcripts in the cyanobacterium Synechocystis 6803. Plant Mol Biol. 1991 May;16(5):891–897. doi: 10.1007/BF00015080. [DOI] [PubMed] [Google Scholar]
  25. Muro-Pastor M. I., Reyes J. C., Florencio F. J. The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria. J Bacteriol. 1996 Jul;178(14):4070–4076. doi: 10.1128/jb.178.14.4070-4076.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mérida A., Candau P., Florencio F. J. In vitro reactivation of in vivo ammonium-inactivated glutamine synthetase from Synechocystis sp. PCC 6803. Biochem Biophys Res Commun. 1991 Dec 16;181(2):780–786. doi: 10.1016/0006-291x(91)91258-e. [DOI] [PubMed] [Google Scholar]
  27. Mérida A., Candau P., Florencio F. J. Regulation of glutamine synthetase activity in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 by the nitrogen source: effect of ammonium. J Bacteriol. 1991 Jul;173(13):4095–4100. doi: 10.1128/jb.173.13.4095-4100.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mérida A., Leurentop L., Candau P., Florencio F. J. Purification and properties of glutamine synthetases from the cyanobacteria Synechocystis sp. strain PCC 6803 and Calothrix sp. strain PCC 7601. J Bacteriol. 1990 Aug;172(8):4732–4735. doi: 10.1128/jb.172.8.4732-4735.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Petersen C. Control of functional mRNA stability in bacteria: multiple mechanisms of nucleolytic and non-nucleolytic inactivation. Mol Microbiol. 1992 Feb;6(3):277–282. doi: 10.1111/j.1365-2958.1992.tb01469.x. [DOI] [PubMed] [Google Scholar]
  30. Ramasubramanian T. S., Wei T. F., Golden J. W. Two Anabaena sp. strain PCC 7120 DNA-binding factors interact with vegetative cell- and heterocyst-specific genes. J Bacteriol. 1994 Mar;176(5):1214–1223. doi: 10.1128/jb.176.5.1214-1223.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Reyes J. C., Florencio F. J. A new type of glutamine synthetase in cyanobacteria: the protein encoded by the glnN gene supports nitrogen assimilation in Synechocystis sp. strain PCC 6803. J Bacteriol. 1994 Mar;176(5):1260–1267. doi: 10.1128/jb.176.5.1260-1267.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Reyes J. C., Florencio F. J. A novel mechanism of glutamine synthetase inactivation by ammonium in the cyanobacterium Synechocystis sp. PCC 6803. Involvement of an inactivating protein. FEBS Lett. 1995 Jun 19;367(1):45–48. doi: 10.1016/0014-5793(95)00544-j. [DOI] [PubMed] [Google Scholar]
  33. Reyes J. C., Florencio F. J. Electron transport controls transcription of the glutamine synthetase gene (glnA) from the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol. 1995 Feb;27(4):789–799. doi: 10.1007/BF00020231. [DOI] [PubMed] [Google Scholar]
  34. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shaw W. V. Chloramphenicol acetyltransferase from chloramphenicol-resistant bacteria. Methods Enzymol. 1975;43:737–755. doi: 10.1016/0076-6879(75)43141-x. [DOI] [PubMed] [Google Scholar]
  36. Stock J. B., Ninfa A. J., Stock A. M. Protein phosphorylation and regulation of adaptive responses in bacteria. Microbiol Rev. 1989 Dec;53(4):450–490. doi: 10.1128/mr.53.4.450-490.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Vega-Palas M. A., Flores E., Herrero A. NtcA, a global nitrogen regulator from the cyanobacterium Synechococcus that belongs to the Crp family of bacterial regulators. Mol Microbiol. 1992 Jul;6(13):1853–1859. doi: 10.1111/j.1365-2958.1992.tb01357.x. [DOI] [PubMed] [Google Scholar]
  38. Vega-Palas M. A., Madueño F., Herrero A., Flores E. Identification and cloning of a regulatory gene for nitrogen assimilation in the cyanobacterium Synechococcus sp. strain PCC 7942. J Bacteriol. 1990 Feb;172(2):643–647. doi: 10.1128/jb.172.2.643-647.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Vioque A. Analysis of the gene encoding the RNA subunit of ribonuclease P from cyanobacteria. Nucleic Acids Res. 1992 Dec 11;20(23):6331–6337. doi: 10.1093/nar/20.23.6331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wagner S. J., Thomas S. P., Kaufman R. I., Nixon B. T., Stevens S. E., Jr The glnA gene of the cyanobacterium Agmenellum quadruplicatum PR-6 is nonessential for ammonium assimilation. J Bacteriol. 1993 Feb;175(3):604–612. doi: 10.1128/jb.175.3.604-612.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wealand J. L., Myers J. A., Hirschberg R. Changes in gene expression during nitrogen starvation in Anabaena variabilis ATCC 29413. J Bacteriol. 1989 Mar;171(3):1309–1313. doi: 10.1128/jb.171.3.1309-1313.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wei T. F., Ramasubramanian T. S., Golden J. W. Anabaena sp. strain PCC 7120 ntcA gene required for growth on nitrate and heterocyst development. J Bacteriol. 1994 Aug;176(15):4473–4482. doi: 10.1128/jb.176.15.4473-4482.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Wei T. F., Ramasubramanian T. S., Pu F., Golden J. W. Anabaena sp. strain PCC 7120 bifA gene encoding a sequence-specific DNA-binding protein cloned by in vivo transcriptional interference selection. J Bacteriol. 1993 Jul;175(13):4025–4035. doi: 10.1128/jb.175.13.4025-4035.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. de Bruijn F. J., Rossbach S., Schneider M., Ratet P., Messmer S., Szeto W. W., Ausubel F. M., Schell J. Rhizobium meliloti 1021 has three differentially regulated loci involved in glutamine biosynthesis, none of which is essential for symbiotic nitrogen fixation. J Bacteriol. 1989 Mar;171(3):1673–1682. doi: 10.1128/jb.171.3.1673-1682.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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