Effect of the Inorganic Nitrogen Source in the Expression of Nitrite Reductase (NirA) in Thermosynechococcus elongatus BP-1

Maite Buxens, María J. Llama, Juan L. Serra^*

Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country (UPV/EHU), Bilbao, Spain.

DOI: 10.4236/aim.2014.415115                

Abstract

Nitrite reductase (NirA, EC 1.7.7.1) from the thermophilic, unicellular, non-N2-fixing cyanobacte-rium Thermosynechococcus elongatus BP-1 has been cloned and expressed in Escherichia coli. Analysis by SDS-PAGE of the pure recombinant protein (His6NirA) showed two protein bands, one of 58 kDa (corresponding to the theoretical His6NirA molecular mass) and another of 44 kDa. Western blotting and mass spectrometry analyses confirmed that the 44 kDa protein resulted from proteolysis of the intact His6NirA, and suggested the existence, at the C-terminal domain of the 58 kDa form, of a region particularly sensible to proteolysis or accessible to proteases. A sample of both forms of His6NirA was used to obtain anti-NirA polyclonal antibodies. These antibodies were used to assess, by SDS-PAGE followed by Western blotting, the in vivo expression of NirA in wild-type cells of T. elongatus BP-1 growing in cultures with nitrate, nitrite or ammonium which were inoculated with cells grown with different nitrogen sources. These analyses revealed that protein bands corresponding to the complete (58 kDa) and truncated (44 kDa) forms of NirA can also be detected in solubilized cells. Moreover, the presence of each of these forms depended on the nitrogen source used to grow cells. Thus, expression of the complete NirA generally predominates in cells growing in medium with nitrate or nitrite. However, the truncated form prevails in cells grown in nitrate or nitrite and then transferred to medium with ammonium. The fact that the patterns of in vivo expression of NirA are different depending on the nitrogen source used possibly relies on a post-translational regulatory mechanism by proteolysis.

Keywords

Cyanobacteria, NirA, Nitrite Reductase Expression, Post-Translational Regulation, Protein Hydrolysis, Thermosynechococcus elongatus BP-1

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	Flores, E. and Herrero, A. (2005) Nitrogen Assimilation and Nitrogen Control in Cyanobacteria. Biochemical Society Transactions, 33, 164-167. http://dx.doi.org/10.1042/BST0330164
[2]	Flores, E., Frías, J.E., Rubio, L.M. and Herrero, A. (2005) Photosynthetic Nitrate Assimilation in Cyanobacteria. Photosynthesis Research, 83, 117-133. http://dx.doi.org/10.1007/s11120-004-5830-9
[3]	Aichi, M., Yoshihara, S., Yamashita, M., Maeda, S., Nagai, K. and Omata, T. (2006) Characterization of the Nitrate-Nitrite Transporter of the Major Facilitator Superfamily (the nrtP Gene Product) from the Cyanobacterium Nostoc punctiforme Strain ATCC 29133. Bioscience Biotechnology and Biochemistry, 70, 2682-2689.http://dx.doi.org/10.1271/bbb.60286
[4]	Ohashi, Y., Shi, W., Takatani, N., Aichi, M., Maeda, S.-i., Watanabe, S., et al. (2011) Regulation of Nitrate Assimilation in Cyanobacteria. Journal of Experimental Botany, 62, 1411-1424. http://dx.doi.org/10.1093/jxb/erq427
[5]	Frías, J.E., Flores, E. and Herrero, A. (1997) Nitrate Assimilation Gene Cluster from the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120. Journal of Bacteriology, 179, 477-486.
[6]	Vega, J.M. and Kamin, H. (1977) Spinach Nitrite Reductase. Purification and Properties of a Siroheme-Containing Iron-Sulphur Enzyme. Journal of Biological Chemistry, 252, 896-909.
[7]	Stroupe, M.E. and Getzoff, E.D. (2009) The Role of Siroheme in Sulfite and Nitrite Reductases. In: Warren, M.J. and Smith, A.G., Eds., Tetrapyrroles: Birth, Life and Death, Springer Science+Business Media, LLC, New York, 375-389.
[8]	Méndez, J.M. and Vega, J.M. (1981) Purification and Molecular Properties of Nitrite Reductase from Anabaena sp. Physiologia Plantarum, 52, 7-14. http://dx.doi.org/10.1111/j.1399-3054.1981.tb06026.x
[9]	Yabuki, Y., Mori, E. and Tamura, G. (1985) Nitrite Reductase in the Cyanobacterium Spirulina platensis. Agricultural and Biological Chemistry, 49, 3061-3062. http://dx.doi.org/10.1271/bbb1961.49.3061
[10]	Guerrero, M.G. and Lara, C. (1987) Assimilation of Inorganic Nitrogen. In: Fay, P. and van Baalen, C., Eds., The Cyanobacteria, Elsevier Science Publishers B.V., Amsterdam, 163-186.
[11]	Arizmendi, J.M. and Serra, J.L. (1990) Purification and Some Properties of the Nitrite Reductase from the Cyanobacterium Phormidium laminosum. Biochimica et Biophysica Acta (BBA), Protein Structure and Molecular Enzymology, 1040, 237-244. http://dx.doi.org/10.1016/0167-4838(90)90082-Q
[12]	Suzuki, I., Kikuchi, H., Nakanishi, S., Fujita, Y., Sugiyama, T. and Omata, T. (1995) A Novel Nitrite Reductase Gene from the Cyanobacterium Plectonema boryanum. Journal of Bacteriology, 177, 6137-6143.
[13]	Flores, E., Guerrero, M.G. and Losada, M. (1980) Short-Term Ammonium Inhibition of Nitrate Utilization by Anacystis nidulans and Other Cyanobacteria. Archives of Microbiology, 128, 137-144. http://dx.doi.org/10.1007/BF00406150
[14]	Arizmendi, J.M., Fresnedo, O., Martínez-Bilbao, M., Alaña, A. and Serra, J.L. (1987) Inorganic Nitrogen Assimilation in the Non-N2-Fixing Cyanobacterium Phormidium laminosum. II. Effect of the Nitrogen Source on the Nitrite Reductase Levels. Physiologia Plantarum, 70, 703-707. http://dx.doi.org/10.1111/j.1399-3054.1987.tb04327.x
[15]	Kobayashi, M., Takatani, N., Tanigawa, M. and Omata, T. (2005) Posttranslational Regulation of Nitrate Assimilation in the Cyanobacterium Synechocystis sp. Strain PCC 6803. Journal of Bacteriology, 187, 498-506.http://dx.doi.org/10.1128/JB.187.2.498-506.2005
[16]	Herrero, A. and Guerrero, M.G. (1986) Regulation of Nitrite Reductase in the Cyanobacterium Anacystis nidulans. Journal of General Microbiology, 132, 2463-2468.
[17]	Tapia, M.I., Llama, M.J. and Serra, J.L. (1996) Regulation of Nitrate Assimilation in the Cyanobacterium Phormidium laminosum. Planta, 198, 24-30. http://dx.doi.org/10.1007/BF00197582
[18]	Puerta-Fernández, E. and Vioque, A. (2011) Hfq Is Required for Optimal Nitrate Assimilation in the Cyanobacterium Anabaena sp. Strain PCC 7120. Journal of Bacteriology, 193, 3546-3555. http://dx.doi.org/10.1128/JB.00254-11
[19]	Frías, J.E. and Flores, E. (2010) Negative Regulation of Expression of the Nitrate Assimilation nirA Operon in the Heterocyst-Forming Cyanobacterium Anabaena sp. Strain PCC 7120. Journal of Bacteriology, 192, 2769-2778. http://dx.doi.org/10.1128/JB.01668-09
[20]	Nakamura, Y., Kaneko, T., Sato, S., Ikeuchi, M., Katoh, H., Sasamoto, S., et al. (2002) Complete Genome Structure of the Thermophilic Cyanobacterium Thermosynechococcus elongatus BP-1. DNA Research, 9, 123-130. http://dx.doi.org/10.1093/dnares/9.4.123
[21]	Buxens, M., Serra, J.L. and Llama, M.J. (2013) Substitution of the Nitrite Reductase of Thermosynechococcus elongatus BP-1 by the Homologous Gene of Phormidium laminosum. Advances in Microbiology, 3, 69-79. http://dx.doi.org/10.4236/aim.2013.36A009
[22]	Stanier, R.Y., Kunisawa, R., Mandel, M. and Cohen-Bazire, G. (1971) Purification and Properties of Unicellular Blue-Green Algae (Order Chroococcales). Bacteriological Reviews, 35, 171-205.
[23]	Bradford, M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding. Analytical Biochemistry, 72, 248-254. http://dx.doi.org/10.1016/0003-2697(76)90527-3
[24]	Mathiesen, R., Bunkenborg, J., Stensballe, A., Jensen, O.N., Welinder, K.G., Bauw, G. (2004) Database-Independent, Database-Dependent, and Extended Interpretation of Peptide Mass Spectra in VEMS V2.0. Proteomics, 4, 2583-2593.http://dx.doi.org/10.1002/pmic.200300792
[25]	Hess, A.V.I. (2007) Digitally-Enhanced Thin-Layer Chromatography: An Inexpensive, New Technique for Qualitative and Quantitative Analysis. Journal of Chemical Education, 84, 842-847. http://dx.doi.org/10.1021/ed084p842
[26]	Serra, J.L., Llama, M.J. and Cadenas, E. (1978) Nitrate Utilization by the Diatom Skeletonema costatum. II. Regulation of Nitrate Uptake. Plant Physiology, 62, 991-994. http://dx.doi.org/10.1104/pp.62.6.991
[27]	Bellissimo, D.B. and Privalle, L.S. (1995) Expression of Spinach Nitrite Reductase in Escherichia coli: Site-Directed Mutagenesis of Predicted Active Site Amino Acids. Archives of Biochemistry and Biophysics, 323, 155-163. http://dx.doi.org/10.1006/abbi.1995.0021
[28]	Ostrowski, J., Wu, J.Y., Rueger, D.C., Miller, B.E., Siegel, L.M. and Kredrich, M. (1989) Characterization of the cysJIH Regions of Salmonella typhimurium and Escherichia coli. Journal of Biological Chemistry, 264, 15726-15737.
[29]	Luque, I., Flores, E. and Herrero, A. (1993) Nitrite Reductase Gene from Synechococcus sp. PCC 7942: Homology between Cyanobacterial and Higher-Plant Nitrite Reductases. Plant Molecular Biology, 21, 1201-1205. http://dx.doi.org/10.1007/BF00023618
[30]	Schnell, R., Sandalova, T., Hellman, U. and Schneider, G. (2005) Siroheme and [Fe4-S4]-Dependent nirA from Mycobacterium tuberculosis Is a Sulphite Reductase with a Covalent Cys-Tyr Bond in the Active Site. Journal of Biological Chemistry, 280, 27319-27328. http://dx.doi.org/10.1074/jbc.M502560200
[31]	Swamy, U., Wang, M., Tripathy, J.N., Kim, S.K., Hirasawa, M., Knaff, D.B., et al. (2005) Structure of Spinach Nitrite Reductase: Implications for Multi-Electron Reactions by the Iron-Sulphur: Siroheme Cofactor. Biochemistry, 44, 16054-16063. http://dx.doi.org/10.1021/bi050981y
[32]	Tischner, R. and Schmidt, A. (1984) Light Mediated Regulation of Nitrate Assimilation in Synechococcus leopoliensis. Archives in Microbiology, 137, 151-154. http://dx.doi.org/10.1007/BF00414457
[33]	Ludwig, M. and Bryant, D.A. (2012) Acclimation of the Global Transcriptome of the Cyanobacterium Synechococcus sp. Strain PCC 7002 to Nutrient Limitations and Different Nitrogen Sources. Frontiers in Microbiology, 3, 145.http://dx.doi.org/10.3389/fmicb.2012.00145