Photosynthesis Research Protocols pp 295-312

Part of the Methods in Molecular Biology book series (MIMB, volume 684) | Cite as

Construction of Gene Interruptions and Gene Deletions in the Cyanobacterium Synechocystis sp. Strain PCC 6803



A series of protocols are presented for the storage, growth, transformation, and characterization of wild type and mutant strains of Synechocystis sp. strain PCC 6803. These protocols include the isolation of genomic DNA and the strategies required for the construction of specific gene interruptions or deletions in this organism. This cyanobacterium has been used widely as a model for photosynthesis research, and the sequence of its genome is available at CyanoBase ( The details provided in this chapter do not assume any previous experience in working with cyanobacteria and are intended to enable new investigators to take advantage of a wide range of gene modification and mutation mapping techniques that have been adapted for use in this system.

Key words

Cell culture CyanoBase DNA isolation Growth curves Mutagenesis Oxygen evolution Synechocystis 


  1. 1.
    Rippka, R., Deruelles, J., Waterbury, J. B., Herdman, M., and Stanier, R. Y. (1979) Generic assignments, strain histories and properties of pure cultures of cyanobacteria. J. Gen. Microbiol. 111, 1–61.Google Scholar
  2. 2.
    Grigorieva, G. and Shestakov, S. (1982) Transformation in the cyanobacterium Synechocystis sp. 6803. FEMS Microbiol. Lett. 13, 367–370.CrossRefGoogle Scholar
  3. 3.
    Yoshihara, S., Geng, X. X., Okamoto, S., et al. (2001) Mutational analysis of genes involved in pilus structure, motility, and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol. 42, 63–73.PubMedCrossRefGoogle Scholar
  4. 4.
    Williams, J. G. K. (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth. Enzymol. 167, 766–778.CrossRefGoogle Scholar
  5. 5.
    Vermaas, W. F. J. (1998) Gene modifications and mutation mapping to study the function of photosystem II. Meth. Enzymol. 297, 293–311.PubMedCrossRefGoogle Scholar
  6. 6.
    Kaneko, T., Sato, S., Kotani, H., et al. (1996) Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Res. 3, 109–136.PubMedCrossRefGoogle Scholar
  7. 7.
    Ikeuchi, M. and Tabata, S. (2001) Synechocystis sp. PCC 6803 – a useful tool in the study of the genetics of cyanobacteria. Photosynth. Res. 70, 73–83.PubMedCrossRefGoogle Scholar
  8. 8.
    MacKinney, G. (1941) Absorption of light by chlorophyll solutions. J. Biol. Chem. 140, 315-322.Google Scholar
  9. 9.
    Allen, J. F. and Holmes, N. G. (1986) Electron transport and redox titration, in: Photosynthesis, Energy Transduction: A Practical Approach (Hipkins, M. F. and Baker, N. R., eds.), IRL Press, Oxford, pp. 103–141.Google Scholar
  10. 10.
    Jansson, C., Debus, R. J., Osiewacz, H. D., Gurevitz, M., and McIntosh, L. (1987) Construction of an obligate photoheterotrophic mutant of the cyanobacterium Synechocystis 6803. Plant Physiol. 85, 1021–1025.PubMedCrossRefGoogle Scholar
  11. 11.
    Chu, H.-A., Nguyen, A. P., and Debus, R. J. (1994) Site-directed photosystem II mutants with perturbed oxygen-evolving properties. 2. Increased binding or photooxidation of manganese in the absence of the extrinsic 33-kDa polypeptide in vivo. Biochemistry 33, 6150–6157.PubMedCrossRefGoogle Scholar
  12. 12.
    Morgan, T. R., Shand, J. A., Clarke, S. M., and Eaton-Rye, J. J. (1998) Specific requirements for cytochrome c-550 and the ­manganese-stabilizing protein in photoautotrophic strains of Synechocystis sp. PCC 6803 with mutations in the domain Gly-351 to Thr-436 of the chlorophyll-binding protein CP47. Biochemistry 41, 14,437–14,449.CrossRefGoogle Scholar
  13. 13.
    Gay, P., Le Coq, D., Steinmetz, M., Ferrari, E., and Hoch, J. A. (1983) Cloning structural gene sacB, which codes for exoenzyme levansucrase of Bacillus subtilis: expression of the gene in Escherichia coli. J. Bacteriol. 153, 1424–1431.PubMedGoogle Scholar
  14. 14.
    Cai, Y. P. and Wolk, C. P. (1990) Use of a conditionally lethal gene in Anabaena sp. Strain PCC-7120 to select for double recombinants and to entrap insertion sequences. J. Bacteriol. 172, 3138–3145.PubMedGoogle Scholar
  15. 15.
    Ermakova-Gerdes, S. and Vermaas, W. (1999) Development of a psbA-less/psbD-less strain of Synechocystis sp. PCC 6803 for simultaneous mutagenesis of the D1 and D2 proteins of photosystem II, in The Phototrophic Prokaryotes (Peschek, G. A. and Loffelhardt, W., eds.), Kluwer, Dordrecht, pp. 51–60.CrossRefGoogle Scholar
  16. 16.
    Kunkel, T. A. (1985) Rapid and efficient site-specific mutagenesis without phenotypic selection. Proc. Natl Acad. Sci. USA 82, 488–492.PubMedCrossRefGoogle Scholar
  17. 17.
    Kunkel, T. A., Roberts, J. D., and Zakour, R. A. (1987) Rapid and efficient site-specific mutagenesis without phenotypic selection. Meth. Enzymol. 154, 367–382.PubMedCrossRefGoogle Scholar
  18. 18.
    Vandeyar, M. A., Weiner, M. P., Hutton, C. J., and Batt, C. A. (1988) A simple and rapid method for the selection of oligodeoxynucleotide-directed mutants. Gene 65, 129–133.PubMedCrossRefGoogle Scholar
  19. 19.
    Vermaas, W., Charite, J., and Eggers, B. (1990) System for site-directed mutagenesis in the psbD1/C operon of Synechocystis sp. PCC 6803, in Current Research in Photosynthesis. Vol. I (Baltscheffsky, M., ed.), Kluwer, Dordrecht, pp. 231–238.Google Scholar
  20. 20.
    Eaton-Rye, J. J. and Vermaas, W. F. J. (1991) Oligonucleotide-directed mutagenesis of psbB, the gene encoding CP47, employing a deletion mutant strain of the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol. Biol. 17, 1165–1177.PubMedCrossRefGoogle Scholar
  21. 21.
    Bentley, F. K., Luo, H., Dilbeck, P., Burnap, R. L., and Eaton-Rye, J. J. (2008) Effects of inactivating psbM and psbT on photodamage and assembly of photosystem II in Synechocystis sp. PCC 6803. Biochemistry 47, 11,637–11,646.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  1. 1.Biochemistry DepartmentUniversity of OtagoDunedinNew Zealand

Personalised recommendations