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. 1996 Jul;178(14):4070–4076. doi: 10.1128/jb.178.14.4070-4076.1996

The NADP+-isocitrate dehydrogenase gene (icd) is nitrogen regulated in cyanobacteria.

M I Muro-Pastor 1, J C Reyes 1, F J Florencio 1
PMCID: PMC178162  PMID: 8763933

Abstract

NADP+-isocitrate dehydrogenase (NADP+-IDH) activity and protein levels in crude extracts from the unicellular cyanobacterium Synechocystis sp. strain PCC 6803 and the filamentous, dinitrogen-fixing Anabaena sp. strain PCC 7120 were determined under different nitrogen conditions. The highest NADP+-IDH activity and protein accumulation were found under dinitrogen-fixing conditions for the Anabaena strain and under nitrogen starvation for Synechocystis sp. PCC 6803. The icd gene that encodes the NADP+-IDH from Synechocystis sp. strain PCC 6803 was cloned by heterologous hybridization with the previously isolated icd gene from Anabaena sp. strain PCC 7120. The two cyanobacterial icd genes show 81% sequence identity and share a typical 44-amino-acid region different from all the other icd genes sequenced so far. The icd gene seems to be essential for Synechocystis growth since attempts to generate a completely segregated icd mutant were unsuccessful. Transcripts of 2.0 and 1.6 kb were detected by Northern (RNA) blot analysis, for the Anabaena and Synecho-cystis icd genes, respectively. Maximal icd mRNA accumulation was reached after 5 It of nitrogen starvation in Synechocystis cells and under dinitrogen-fixing conditions in Anabaena cells. Primer extension analysis showed that the structure of the Synechocystis icd gene promoter resembles those of the NtcA-regulated promoters. In addition, mobility shift assays demonstrated that purified Synechocystis NtcA protein binds to the promoter of the icd gene. All these data suggest that the expression of the icd gene from Synechocystis sp. strain PCC 6803 may be subjected to nitrogen control mediated by the positively acting regulatory protein NtcA.

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Selected References

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  1. Bauer C. C., Scappino L., Haselkorn R. Growth of the cyanobacterium Anabaena on molecular nitrogen: NifJ is required when iron is limited. Proc Natl Acad Sci U S A. 1993 Oct 1;90(19):8812–8816. doi: 10.1073/pnas.90.19.8812. [DOI] [PMC free article] [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: 10.1006/abio.1976.9999. [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. Chastain C. J., Brusca J. S., Ramasubramanian T. S., Wei T. F., Golden J. W. A sequence-specific DNA-binding factor (VF1) from Anabaena sp. strain PCC 7120 vegetative cells binds to three adjacent sites in the xisA upstream region. J Bacteriol. 1990 Sep;172(9):5044–5051. doi: 10.1128/jb.172.9.5044-5051.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Cupp J. R., McAlister-Henn L. Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae. J Biol Chem. 1992 Aug 15;267(23):16417–16423. [PubMed] [Google Scholar]
  7. Cupp J. R., McAlister-Henn L. NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae. J Biol Chem. 1991 Nov 25;266(33):22199–22205. [PubMed] [Google Scholar]
  8. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eikmanns B. J., Rittmann D., Sahm H. Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme. J Bacteriol. 1995 Feb;177(3):774–782. doi: 10.1128/jb.177.3.774-782.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Elhai J., Wolk C. P. A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene. 1988 Aug 15;68(1):119–138. doi: 10.1016/0378-1119(88)90605-1. [DOI] [PubMed] [Google Scholar]
  11. Floriano B., Herrero A., Flores E. Isolation of arginine auxotrophs, cloning by mutant complementation, and sequence analysis of the argC gene from the cyanobacterium Anabaena species PCC 7120. Mol Microbiol. 1992 Aug;6(15):2085–2094. doi: 10.1111/j.1365-2958.1992.tb01381.x. [DOI] [PubMed] [Google Scholar]
  12. Forchhammer K., Tandeau de Marsac N. Phosphorylation of the PII protein (glnB gene product) in the cyanobacterium Synechococcus sp. strain PCC 7942: analysis of in vitro kinase activity. J Bacteriol. 1995 Oct;177(20):5812–5817. doi: 10.1128/jb.177.20.5812-5817.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. 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]
  15. Haselbeck R. J., Colman R. F., McAlister-Henn L. Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase. Biochemistry. 1992 Jul 14;31(27):6219–6223. doi: 10.1021/bi00142a007. [DOI] [PubMed] [Google Scholar]
  16. Haselbeck R. J., McAlister-Henn L. Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. J Biol Chem. 1991 Feb 5;266(4):2339–2345. [PubMed] [Google Scholar]
  17. Holland D., Wolk C. P. Identification and characterization of hetA, a gene that acts early in the process of morphological differentiation of heterocysts. J Bacteriol. 1990 Jun;172(6):3131–3137. doi: 10.1128/jb.172.6.3131-3137.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Huh T. L., Ryu J. H., Huh J. W., Sung H. C., Oh I. U., Song B. J., Veech R. L. Cloning of a cDNA encoding bovine mitochondrial NADP(+)-specific isocitrate dehydrogenase and structural comparison with its isoenzymes from different species. Biochem J. 1993 Jun 15;292(Pt 3):705–710. doi: 10.1042/bj2920705. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hurley J. H., Thorsness P. E., Ramalingam V., Helmers N. H., Koshland D. E., Jr, Stroud R. M. Structure of a bacterial enzyme regulated by phosphorylation, isocitrate dehydrogenase. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8635–8639. doi: 10.1073/pnas.86.22.8635. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Ishii A., Ochiai T., Imagawa S., Fukunaga N., Sasaki S., Minowa O., Mizuno Y., Shiokawa H. Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions. J Biochem. 1987 Dec;102(6):1489–1498. doi: 10.1093/oxfordjournals.jbchem.a122196. [DOI] [PubMed] [Google Scholar]
  21. Ishii A., Suzuki M., Sahara T., Takada Y., Sasaki S., Fukunaga N. Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol. 1993 Nov;175(21):6873–6880. doi: 10.1128/jb.175.21.6873-6880.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Jin S., Sonenshein A. L. Identification of two distinct Bacillus subtilis citrate synthase genes. J Bacteriol. 1994 Aug;176(15):4669–4679. doi: 10.1128/jb.176.15.4669-4679.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Jin S., Sonenshein A. L. Transcriptional regulation of Bacillus subtilis citrate synthase genes. J Bacteriol. 1994 Aug;176(15):4680–4690. doi: 10.1128/jb.176.15.4680-4690.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kolb A., Busby S., Buc H., Garges S., Adhya S. Transcriptional regulation by cAMP and its receptor protein. Annu Rev Biochem. 1993;62:749–795. doi: 10.1146/annurev.bi.62.070193.003533. [DOI] [PubMed] [Google Scholar]
  25. LaPorte D. C., Thorsness P. E., Koshland D. E., Jr Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment. J Biol Chem. 1985 Sep 5;260(19):10563–10568. [PubMed] [Google Scholar]
  26. Labarre J., Chauvat F., Thuriaux P. Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803. J Bacteriol. 1989 Jun;171(6):3449–3457. doi: 10.1128/jb.171.6.3449-3457.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  28. Lakshmi T. M., Helling R. B. Selection for citrate synthase deficiency in icd mutants of Escherichia coli. J Bacteriol. 1976 Jul;127(1):76–83. doi: 10.1128/jb.127.1.76-83.1976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Leyland M. L., Kelly D. J. Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii. Eur J Biochem. 1991 Nov 15;202(1):85–93. doi: 10.1111/j.1432-1033.1991.tb16347.x. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. McDermott T. R., Kahn M. L. Cloning and mutagenesis of the Rhizobium meliloti isocitrate dehydrogenase gene. J Bacteriol. 1992 Jul;174(14):4790–4797. doi: 10.1128/jb.174.14.4790-4797.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. 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]
  33. 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]
  34. Miyazaki K., Eguchi H., Yamagishi A., Wakagi T., Oshima T. Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8. Appl Environ Microbiol. 1992 Jan;58(1):93–98. doi: 10.1128/aem.58.1.93-98.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Mulligan M. E., Haselkorn R. Nitrogen fixation (nif) genes of the cyanobacterium Anabaena species strain PCC 7120. The nifB-fdxN-nifS-nifU operon. J Biol Chem. 1989 Nov 15;264(32):19200–19207. [PubMed] [Google Scholar]
  37. Muro-Pastor M. I., Florencio F. J. NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene. J Bacteriol. 1994 May;176(9):2718–2726. doi: 10.1128/jb.176.9.2718-2726.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Muro-Pastor M. I., Florencio F. J. Purification and properties of NADP-isocitrate dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC 6803. Eur J Biochem. 1992 Jan 15;203(1-2):99–105. doi: 10.1111/j.1432-1033.1992.tb19833.x. [DOI] [PubMed] [Google Scholar]
  39. 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]
  40. Pearce J., Leach C. K., Carr N. G. The incomplete tricarboxylic acid cycle in the blue-green alga Anabaena variabilis. J Gen Microbiol. 1969 Mar;55(3):371–378. doi: 10.1099/00221287-55-3-371. [DOI] [PubMed] [Google Scholar]
  41. 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]
  42. Reeves H. C., Daumy G. O., Lin C. C., Houston M. NADP + -specific isocitrate dehydrogenase of Escherichia coli. I. Purification and characterization. Biochim Biophys Acta. 1972 Jan 20;258(1):27–39. doi: 10.1016/0005-2744(72)90964-3. [DOI] [PubMed] [Google Scholar]
  43. 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]
  44. 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]
  45. Shorrosh B. S., Dixon R. A. Molecular characterization and expression of an isocitrate dehydrogenase from alfalfa (Medicago sativa L). Plant Mol Biol. 1992 Dec;20(5):801–807. doi: 10.1007/BF00027151. [DOI] [PubMed] [Google Scholar]
  46. Stanier R. Y., Cohen-Bazire G. Phototrophic prokaryotes: the cyanobacteria. Annu Rev Microbiol. 1977;31:225–274. doi: 10.1146/annurev.mi.31.100177.001301. [DOI] [PubMed] [Google Scholar]
  47. Suzuki M., Sahara T., Tsuruha J., Takada Y., Fukunaga N. Differential expression in Escherichia coli of the Vibrio sp. strain ABE-1 icdI and icdII genes encoding structurally different isocitrate dehydrogenase isozymes. J Bacteriol. 1995 Apr;177(8):2138–2142. doi: 10.1128/jb.177.8.2138-2142.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. 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]
  49. 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]
  50. 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]
  51. 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]

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