Aspartate and glutamate are two key amino acids used in biosynthesis of many amino acids that play vital role in cellular metabolism. Aspartate aminotransferases (AspATs) are required for channelling nitrogen (N) between Glu and Asp in all life forms. Biochemical and genetic characterization of AspATs have been lacking in N-fixing cyanobacteria. In this report, five putative AspAT genes (, , , and ) were identified in the N-fixing heterocystous cyanobacterium sp. PCC 7120. Five recombinant C-terminal hexahistidine-tagged AspATs (AspAT-H) were overexpressed in and purified to homogeneity. Biochemical analysis demonstrated that these five putative AspATs have authentic AspAT activity using aspartate as an amino donor. However, the enzymic activities of the five AspATs differed . Alr4853-H showed the highest AspAT activity, while the enzymic activity for the other four AspATs ranged from 6.5 to 53.7 % activity compared to Alr4853 (100 %). Genetic characterization of the five AspAT genes was also performed by inactivating each individual gene. All of the five AspAT knockout mutants exhibited reduced diazotrophic growth, and was further identified to be a Fox gene (requiring fixed N for growth in the presence of oxygen). Four out of five P transcriptional fusions were constitutively expressed in both diazotrophic and nitrate-dependent growth conditions. Quantitative reverse transcriptase PCR showed that expression was increased by 2.3-fold after 24 h of N deprivation. Taken together, these findings add to our understanding of the role of AspATs in N-fixing within heterocystous cyanobacteria.


Article metrics loading...

Loading full text...

Full text loading...



  1. Alfano J. R., Kahn M. L. (1993). Isolation and characterization of a gene coding for a novel aspartate aminotransferase from Rhizobium meliloti . J Bacteriol 175, 41864196[PubMed] [Google Scholar]
  2. Allen M. B., Arnon D. I. (1955). Studies on nitrogen-fixing blue-green algae. I. Growth and nitrogen fixation by Anabaena cylindrica Lemm. Plant Physiol 30, 366372 [View Article][PubMed] [Google Scholar]
  3. Chen K., Gu L., Xiang X., Lynch M., Zhou R. (2012). Identification and characterization of five intramembrane metalloproteases in Anabaena variabilis . J Bacteriol 194, 61056115 [View Article][PubMed] [Google Scholar]
  4. Chen K., Xu X., Gu L., Hildreth M., Zhou R. (2015). Simultaneous gene inactivation and promoter reporting in cyanobacteria. Appl Microbiol Biotechnol 99, 17791793 [View Article][PubMed] [Google Scholar]
  5. de la Torre F., De Santis L., Suárez M. F., Crespillo R., Cánovas F. M. (2006). Identification and functional analysis of a prokaryotic-type aspartate aminotransferase: implications for plant amino acid metabolism. Plant J 46, 414425 [View Article][PubMed] [Google Scholar]
  6. de la Torre F., Suárez M. F., Santis L., Cánovas F. M. (2007). The aspartate aminotransferase family in conifers: biochemical analysis of a prokaryotic-type enzyme from maritime pine. Tree Physiol 27, 12831291 [View Article][PubMed] [Google Scholar]
  7. de la Torre F., El-Azaz J., Avila C., Cánovas F. M. (2014). Deciphering the role of aspartate and prephenate aminotransferase activities in plastid nitrogen metabolism. Plant Physiol 164, 92104 [View Article][PubMed] [Google Scholar]
  8. Dereeper A., Guignon V., Blanc G., Audic S., Buffet S., Chevenet F., Dufayard J. F., Guindon S., Lefort V., other authors. (2008). Phylogeny.fr: robust phylogenetic analysis for the non-specialist. Nucleic Acids Res 36 (Web Server), W465W469 [View Article][PubMed] [Google Scholar]
  9. Elhai J., Wolk C. P. (1988). Conjugal transfer of DNA to cyanobacteria. Methods Enzymol 167, 747754 [View Article][PubMed] [Google Scholar]
  10. Elhai J., Vepritskiy A., Muro-Pastor A. M., Flores E., Wolk C. P. (1997). Reduction of conjugal transfer efficiency by three restriction activities of Anabaena sp. strain PCC 7120. J Bacteriol 179, 19982005[PubMed] [Google Scholar]
  11. Fan Q., Huang G., Lechno-Yossef S., Wolk C. P., Kaneko T., Tabata S. (2005). Clustered genes required for synthesis and deposition of envelope glycolipids in Anabaena sp. strain PCC 7120. Mol Microbiol 58, 227243 [View Article][PubMed] [Google Scholar]
  12. Flaherty B. L., Van Nieuwerburgh F., Head S. R., Golden J. W. (2011). Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation. BMC Genomics 12, 332 [View Article][PubMed] [Google Scholar]
  13. Graindorge M., Giustini C., Jacomin A. C., Kraut A., Curien G., Matringe M. (2010). Identification of a plant gene encoding glutamate/aspartate-prephenate aminotransferase: the last homeless enzyme of aromatic amino acids biosynthesis. FEBS Lett 584, 43574360 [View Article][PubMed] [Google Scholar]
  14. Halfmann C., Gu L. P., Zhou R. B. (2014). Engineering cyanobacteria for the production of a cyclic hydrocarbon fuel from CO2 and H2O. Green Chem 16, 31753185 [View Article] [Google Scholar]
  15. Inoue K., Kuramitsu S., Okamoto A., Hirotsu K., Higuchi T., Kagamiyama H. (1991). Site-directed mutagenesis of Escherichia coli aspartate aminotransferase: role of Tyr70 in the catalytic processes. Biochemistry 30, 77967801 [View Article][PubMed] [Google Scholar]
  16. Jäger J., Moser M., Sauder U., Jansonius J. N. (1994). Crystal structures of Escherichia coli aspartate aminotransferase in two conformations. Comparison of an unliganded open and two liganded closed forms. J Mol Biol 239, 285305 [View Article][PubMed] [Google Scholar]
  17. Jansonius J. N., Vincent M. G. (1987). Structural basis for catalysis by aspartate aminotransferase. In Biological Macromolecules and Assemblies, vol. 3, pp. 187285. Edited by F. A. Jurnak & A. McPherson.New York: John Wiley & Sons. [Google Scholar]
  18. Jensen R. A., Gu W. (1996). Evolutionary recruitment of biochemically specialized subdivisions of Family I within the protein superfamily of aminotransferases. J Bacteriol 178, 21612171[PubMed] [Google Scholar]
  19. Jüttner F. (1983). 14C-labeled metabolites in heterocysts and vegetative cells of Anabaena cylindrica filaments and their presumptive function as transport vehicles of organic carbon and nitrogen. J Bacteriol 155, 628633[PubMed] [Google Scholar]
  20. Kim H., Ikegami K., Nakaoka M., Yagi M., Shibata H., Sawa Y. (2003a). Characterization of aspartate aminotransferase from the cyanobacterium Phormidium lapideum . Biosci Biotechnol Biochem 67, 490498 [View Article][PubMed] [Google Scholar]
  21. Kim H., Nakaoka M., Yagi M., Ashida H., Hamada K., Shibata H., Sawa Y. (2003b). Cloning, structural analysis and expression of the gene encoding aspartate aminotransferase from the thermophilic cyanobacterium Phormidium lapideum . J Biosci Bioeng 95, 421424 [View Article][PubMed] [Google Scholar]
  22. Lechno-Yossef S., Fan Q., Wojciuch E., Wolk C. P. (2011). Identification of ten Anabaena sp. genes that under aerobic conditions are required for growth on dinitrogen but not for growth on fixed nitrogen. J Bacteriol 193, 34823489 [View Article][PubMed] [Google Scholar]
  23. Lehninger A. L., Nelson D. L., Cox M. M. (2000). Lehninger Principles of Biochemistry, 3rd edn., New York: Worth Publishers. [Google Scholar]
  24. Maeda H., Yoo H., Dudareva N. (2011). Prephenate aminotransferase directs plant phenylalanine biosynthesis via arogenate. Nat Chem Biol 7, 1921 [View Article][PubMed] [Google Scholar]
  25. Malcolm B. A., Kirsch J. F. (1985). Site-directed mutagenesis of aspartate aminotransferase from E. coli . Biochem Biophys Res Commun 132, 915921 [View Article][PubMed] [Google Scholar]
  26. Martín-Figueroa E., Navarro F., Florencio F. J. (2000). The GS-GOGAT pathway is not operative in the heterocysts. Cloning and expression of glsF gene from the cyanobacterium Anabaena sp. PCC 7120. FEBS Lett 476, 282286 [View Article][PubMed] [Google Scholar]
  27. Miesak B. H., Coruzzi G. M. (2002). Molecular and physiological analysis of Arabidopsis mutants defective in cytosolic or chloroplastic aspartate aminotransferase. Plant Physiol 129, 650660 [View Article][PubMed] [Google Scholar]
  28. Muriana F. J., Alvarez-Ossorio M. C., Relimpio A. M. (1991). Purification and characterization of aspartate aminotransferase from the halophile archaebacterium Haloferax mediterranei . Biochem J 278, 149154[PubMed] [Google Scholar]
  29. Okamoto A., Kato R., Masui R., Yamagishi A., Oshima T., Kuramitsu S. (1996). An aspartate aminotransferase from an extremely thermophilic bacterium, Thermus thermophilus HB8. J Biochem 119, 135144 [View Article][PubMed] [Google Scholar]
  30. Pernil R., Herrero A., Flores E. (2010). Catabolic function of compartmentalized alanine dehydrogenase in the heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120. J Bacteriol 192, 51655172 [View Article][PubMed] [Google Scholar]
  31. Pfaffl M. W. (2001). A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29, e45 [View Article][PubMed] [Google Scholar]
  32. Picossi S., Valladares A., Flores E., Herrero A. (2004). Nitrogen-regulated genes for the metabolism of cyanophycin, a bacterial nitrogen reserve polymer: expression and mutational analysis of two cyanophycin synthetase and cyanophycinase gene clusters in heterocyst-forming cyanobacterium Anabaena sp. PCC 7120. J Biol Chem 279, 1158211592 [View Article][PubMed] [Google Scholar]
  33. Pinto F., Pacheco C. C., Ferreira D., Moradas-Ferreira P., Tamagnini P. (2012). Selection of suitable reference genes for RT-qPCR analyses in cyanobacteria. PLoS One 7, e34983 [View Article][PubMed] [Google Scholar]
  34. Schultz C. J., Coruzzi G. M. (1995). The aspartate aminotransferase gene family of Arabidopsis encodes isoenzymes localized to three distinct subcellular compartments. Plant J 7, 6175 [View Article][PubMed] [Google Scholar]
  35. Schultz C. J., Hsu M., Miesak B., Coruzzi G. M. (1998). Arabidopsis mutants define an in vivo role for isoenzymes of aspartate aminotransferase in plant nitrogen assimilation. Genetics 149, 491499[PubMed] [Google Scholar]
  36. Siregar I. Z., Yunanto T. (2010). The genetics of glutamate oxaloacetate transaminase (GOT) in Pinus merkusii Jungh. et de Vriese. Biodiversitas 11, 58 [View Article] [Google Scholar]
  37. Sung M. H., Tanizawa K., Tanaka H., Kuramitsu S., Kagamiyama H., Hirotsu K., Okamoto A., Higuchi T., Soda K. (1991). Thermostable aspartate aminotransferase from a thermophilic Bacillus species. Gene cloning, sequence determination, and preliminary x-ray characterization. J Biol Chem 266, 25672572[PubMed] [Google Scholar]
  38. Thomas J., Meeks J. C., Wolk C. P., Shaffer P. W., Austin S. M. (1977). Formation of glutamine from [13N]ammonia, [13N]dinitrogen, and [14C]glutamate by heterocysts isolated from Anabaena cylindrica . J Bacteriol 129, 15451555[PubMed] [Google Scholar]
  39. Zhou R., Kroos L. (2004). BofA protein inhibits intramembrane proteolysis of pro-sigmaK in an intercompartmental signaling pathway during Bacillus subtilis sporulation. Proc Natl Acad Sci U S A 101, 63856390 [View Article][PubMed] [Google Scholar]
  40. Zhou R., Wolk C. P. (2002). Identification of an akinete marker gene in Anabaena variabilis . J Bacteriol 184, 25292532 [View Article][PubMed] [Google Scholar]

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error