Skip to main content
SpringerLink
Log in

Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress

  • Research
  • Published:
Molecular Biotechnology Aims and scope Submit manuscript

Abstract

Superoxide dismutase (SOD) detoxifies cell-toxic superoxide radicals and constitutes an important component of antioxidant machinery in aerobic organisms, including cyanobacteria. The iron-containing SOD (SodB) is one of the most abundant soluble proteins in the cytosol of the nitrogen-fixing cyanobacterium Nostoc punctiforme ATCC 29133, and therefore, we investigated its biochemical properties and response to oxidative stress. The putative SodB-encoding open reading frame Npun_R6491 was cloned and overexpressed in Escherichia coli as a C-terminally hexahistidine-tagged protein. The purified recombinant protein had a SodB specific activity of 2560 ± 48 U/mg protein at pH 7.8 and was highly thermostable. The presence of a characteristic iron absorption peak at 350 nm, and its sensitivity to H2O2 and azide, confirmed that the SodB is an iron-containing SOD. Transcript level of SodB in nitrogen-fixing cultures of N. punctiforme decreased considerably (threefold) after exposure to an oxidative stress-generating herbicide methyl viologen for 4 h. Furthermore, in-gel SOD activity analysis of such cultures grown at increasing concentrations of methyl viologen also showed a loss of SodB activity. These results suggest that SodB is not the primary scavenger of superoxide radicals induced by methyl viologen in N. punctiforme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  1. Latifi, A., Ruiz, M., & Zhang, C. C. (2009). Oxidative stress in cyanobacteria. FEMS Microbiology Reviews, 33, 258–278.

    Article  CAS  Google Scholar 

  2. Simon, F. W., Grzebyk, D., & Schofield, O. (2005). The role and evolution of superoxide dismutases in algae. Journal of Phycology, 41, 453–465.

    Article  Google Scholar 

  3. Pilon, M., Ravet, K., & Tapken, W. (2011). The biogenesis and physiological function of chloroplast superoxide dismutases. Biochimica and Biophyica Acta, 1807, 980–998.

    Google Scholar 

  4. Lancaster, V. L., LoBrutto, R., Selvaraj, F. M., & Blankenship, R. E. (2004). A cambialistic superoxide dismutase in the thermophilic photosynthetic bacterium Chloroflexus aurantiacus. Journal of Bacteriology, 186, 3408–3414.

    Article  CAS  Google Scholar 

  5. Priya, B., Premanandh, J., Dhanalakshmi, R. T., Seethalakshmi, T., Uma, L., Prabaharan, D., et al. (2007). Comparative analysis of cyanobacterial superoxide dismutases to discriminate canonical form. BMC Genomics, 8, 435.

    Article  Google Scholar 

  6. Kim, J. H., & Suh, K. H. (2005). Light-dependent expression of superoxide dismutase from cyanobacterium Synechocystis sp. strain PCC 6803. Archives of Microbiology, 183, 218–223.

    Article  CAS  Google Scholar 

  7. Ke, W. T., Dai, G. Z., Jiang, H. B., Zhang, R., & Qiu, B. S. (2014). Essential roles of iron superoxide dismutase in photoautotrophic growth of Synechocystis sp. PCC 6803 and heterogeneous expression of marine Synechococcus sp. CC9311 copper/zinc superoxide dismutase within its sodB knockdown mutant. Microbiology, 160, 228–241.

    Article  CAS  Google Scholar 

  8. Regelsberger, G., Laaha, U., Dietmann, D., Ruker, F., Caninin, A., Caiola, M. G., et al. (2004). The iron superoxide dismutase from the filamentous cyanobacterium Nostoc PCC 7120: Localization, overexpression, and biochemical characterization. Journal of Biological Chemistry, 279, 44384–44393.

    Article  CAS  Google Scholar 

  9. Li, T., Huang, X., Zhou, R., Liu, Y., Li, B., Nomura, C., et al. (2002). Differential expression and localization of Mn and Fe superoxide dismutases in the heterocystous cyanobacterium Anabaena PCC 7120. Journal of Bacteriology, 184, 5096–5103.

    Article  CAS  Google Scholar 

  10. Bhattacharya, J., GhoshDastidar, K., Chatterjee, A., Majee, M., & Lahiri Majumder, A. (2004). Synechocystis Fe superoxide dismutase gene confers oxidative stress tolerance to Escherichia coli. Biochemical and Biophysical Research Communication, 316, 540–544.

    Article  CAS  Google Scholar 

  11. Moirangthem, L. D., Bhattacharya, S., Stensjö, K., Lindblad, P., & Bhattacharya, J. (2014). A high constitutive catalase activity confers resistance to methyl viologen-promoted oxidative stress in a mutant of the cyanobacterium Nostoc punctiforme ATCC 29133. Applied Microbiology and Biotechnology, 98, 3809–3818.

    Article  CAS  Google Scholar 

  12. Ow, S. Y., Noirel, J., Cardona, T., Taton, A., Lindblad, P., Stensjö, K., et al. (2009). Quantitative overview of N2 fixation in Nostoc punctiforme ATCC 29133 through cellular enrichments and iTRAQ shotgun proteomics. Journal of Proteome Research, 8, 187–189.

    Article  CAS  Google Scholar 

  13. Raghavan, P. S., Rajaram, H., & Apte, S. K. (2011). Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC 7120. Plant Molecular Biology, 77, 407–417.

    Article  CAS  Google Scholar 

  14. Thomas, D. J., Avenson, T. J., Thomas, J. B., & Herbert, S. K. (1998). A cyanobacterium lacking iron superoxide dismutase is sensitized to oxidative stress induced with methyl viologen but is not sensitized to oxidative stress induced with norflurazon. Plant Physiology, 116, 1593–1602.

    Article  CAS  Google Scholar 

  15. Ushimaru, T., Nishiyama, Y., Hayashi, H., & Murata, N. (2002). No coordinated transcriptional regulation of the Sod-Kat antioxidative system in Synechocystis sp. PCC 6803. Journal of Plant Physiology, 159, 805–807.

    Article  CAS  Google Scholar 

  16. Shirkey, B., Kovarcik, D. P., Wright, D. J., Wilmoth, G., Prickett, T. F., Helm, R. F., et al. (2000). Active Fe-containing superoxide dismutase and abundant sodF mRNA in Nostoc commune (cyanobacteria) after years of dessication. Journal of Bacteriology, 182, 189–197.

    Article  CAS  Google Scholar 

  17. Ismaiel, M. M. S., El-Ayouty, Y. M., Loewen, P. C., & Piercey-Normore, M. D. (2014). Characterization of the iron-containing superoxide dismutase and its response to stress in cyanobacterium Spirulina (Arthrospira) platensis. Journal of Applied Phycology, 26, 1649–1658.

    Article  CAS  Google Scholar 

  18. Hunsucker, S. W., Klage, K., Saughter, S. M., Potts, M., & Helm, R. F. (2004). A preliminary investigation of the Nostoc punctiforme proteome. Biochemical and Biophysical Research Communication, 317, 1121–1127.

    Article  CAS  Google Scholar 

  19. Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., & Stanier, R. Y. (1979). Generic assignments, strain histories and properties of pure cultures of cyanobacteria. Journal of General Microbiology, 111, 1–61.

    Article  Google Scholar 

  20. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., & Lipman, D. J. (1990). Basic local alignment search tool. Journal of Molecular Biology, 215, 403–410.

    Article  CAS  Google Scholar 

  21. Rice, P., Longden, I., & Bleasby, A. (2000). EMBOSS: The European Molecular Biology Open Software Suite. Trends in Genetics, 16, 76–277.

    Article  Google Scholar 

  22. Mazur, B. J., Rice, D., & Haselkorn, R. (1980). Identification of blue green algal nitrogen fixation genes by using heterologous DNA hybridization probes. Proceedings of National Academy of Sciences USA, 77, 186–190.

    Article  CAS  Google Scholar 

  23. Bradford, M. 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.

    Article  CAS  Google Scholar 

  24. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–685.

    Article  CAS  Google Scholar 

  25. Beauchamp, C., & Fridovich, I. (1971). Superoxide dismutase: Improved assays and an assay applicable to acrylamide gels. Analytical Biochemistry, 44, 276–287.

    Article  CAS  Google Scholar 

  26. Agervald, Å., Stensjö, K., Holmqvist, M., & Lindblad, P. (2008). Transcription of the extended hyp-operon in Nostoc sp. strain PCC 7120. BMC Microbiology, 8, 69.

    Article  Google Scholar 

  27. Campbell, W. S., & Laudenbach, D. E. (1995). Characterization of four superoxide dismutase genes from a filamentous cyanobacterium. Journal of Bacteriology, 177, 964–972.

    CAS  Google Scholar 

  28. Babbs, C. F., Pham, J. A., & Coolbaugh, R. C. (1989). Lethal hydroxyl radical production in paraquat-treated plants. Plant Physiology, 90, 1267–1270.

    Article  CAS  Google Scholar 

  29. Agervald, Å., Baebprasert, W., Zhang, X., Incharoensakdi, A., Lindblad, P., & Stensjö, K. (2010). The CyAbrB transcription factor CalA regulates the iron superoxide dismutase in Nostoc sp. strain PCC 7120. Environmental Microbiology, 12, 2826–2837.

    CAS  Google Scholar 

  30. Ekman, M., Sandh, G., Nenninger, A., Oliveira, P., & Stensjö, K. (2014). Cellular and functional specificity among ferritin-like proteins in the multicellular cyanobacterium Nostoc punctiforme. Environmental Microbiology, 16, 829–844.

    Article  CAS  Google Scholar 

Download references

Acknowledgments

MLD, RV and KSI acknowledge the financial support from Department of Science and Technology (DST), University Grant Commission (UGC) and Department of Biotechnology (DBT), Government of India, respectively. KS and PL acknowledge the financial support by the Swedish Energy Agency and KA Wallenberg Foundation. JB is grateful to Prof. Conrad W. Mullineaux, Queen Mary University of London, for providing pET-26b plasmid and Dr. Sudeshna Bhattacharya for her technical support. The authors also acknowledge DST for providing financial support in the form of a project, and DBT for Bioinformatics centre and research facility in the form of State Biotech hub.

Author information

Authors and Affiliations

  1. Department of Biotechnology, Mizoram University, PB No. 190, Aizawl, Mizoram, 796004, India

    Lakshmipyari Devi Moirangthem, Kalibulla Syed Ibrahim, Rebecca Vanlalsangi & Jyotirmoy Bhattacharya

  2. Department of Chemistry- Ångström Laboratory, Science for Life Laboratory, Uppsala University, Box 523, 751 20, Uppsala, Sweden

    Karin Stensjö & Peter Lindblad

Authors
  1. Lakshmipyari Devi Moirangthem
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Kalibulla Syed Ibrahim
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Rebecca Vanlalsangi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Karin Stensjö
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Peter Lindblad
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Jyotirmoy Bhattacharya
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jyotirmoy Bhattacharya.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Moirangthem, L.D., Ibrahim, K.S., Vanlalsangi, R. et al. Molecular Cloning and Biochemical Characterization of the Iron Superoxide Dismutase from the Cyanobacterium Nostoc punctiforme ATCC 29133 and Its Response to Methyl Viologen-Induced Oxidative Stress. Mol Biotechnol 57, 1003–1009 (2015). https://doi.org/10.1007/s12033-015-9894-x

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12033-015-9894-x

Keywords

Search

Navigation