Plant Molecular Biology

, Volume 30, Issue 2, pp 243–254 | Cite as

Random chemical mutagenesis of a specific psbDI region coding for a lumenal loop of the D2 protein of photosystem II in Synechocystis sp. PCC 6803

  • Svetlana Ermakova-Gerdes
  • Sergey Shestakov
  • Wim Vermaas
Research article


To identify amino acid residues of the D2 protein that are critical for functional photosystem II (PS II), sodium bisulfite was utilized for in vitro random mutagenesis of the psbDI gene from Synechocystis sp. PCC 6803. Sodium bisulfite reacts specifically with cytosine in single-stranded regions of DNA and does not attack double-stranded DNA. Using a hybrid plasmid that was single-stranded in the region to be mutagenized and that was double-stranded elsewhere, mutations were targeted to a specific psbDI region coding for the lumenal A-B loop of the D2 protein. Several mutants were isolated with a total of 15 different amino acid changes in the loop. The majority of these mutations did not result in a loss of photoautotrophic growth or in significantly altered PS II function. However, mutation of Glu-69 to Lys, Ser-79 to Phe, and Ser-88 to Phe were found to influence photosystem II activity; the importance of the latter two residues for proper PS II function was unexpected. Cells carrying the double mutation S79F/S88F in D2 did not grow photoautotrophically and had no functionally active PS II centers. The single mutant S79F was also incapable of photoautrophic growth, but displayed reasonably stable oxygen evolution, while PS II function in the single mutant S88F appeared to be close to normal. Because of the more pronounced phenotype of the S79F/S88F strain as compared to the single mutants, both Ser residues appear to affect stable assembly and function of the PS II complex. The mechanism by which the S79F mutant loses photoautotrophic growth remains to be established. However, these results show the potential of targeted random mutagenesis to identify functionally important residues in selected regions of proteins.

Key words

Cyanobacteria photosynthesis random mutagenesis sodium bisulfite thylakoids 


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  1. 1.
    Allen JP, Feher G, Yeates TO, Komiya H, Rees DC: Structure of the reaction center from Rhodobacter sphaeroides R-26: The cofactors. Proc Natl Acad Sci USA 84: 6162–6166 (1987).Google Scholar
  2. 2.
    Burnap RL, Shen J-R, Jursinic PA, Inoue Y, Sherman LA: Oxygen yield and thermoluminescence characteristics of a cyanobacterium lacking the manganese-stabilizing protein of Photosystem II. Biochemistry 31: 7404–7410 (1992).Google Scholar
  3. 3.
    Chu H-A, Nguyen AP, Debus RJ: Amino acid residues that influence the binding of manganese or calcium to Photosystem II. 1. The lumenal inter-helical domains of the D1 polypeptide. Biochemistry 34: 5839–5858 (1995).Google Scholar
  4. 4.
    Chu H-A, Nguyen AP, Debus RJ: Amino acid residues that influence the binding of manganese or calcium to Photosystem II. 2. The carboxy-terminal domain of the D1 polypeptide. Biochemistry 34: 5859–5882 (1995).Google Scholar
  5. 5.
    Chu H-A, Nguyen AP, Debus RJ: Site-directed photosystem II mutants with perturbed oxygen-evolving properties. 1. Instability or inefficient assembly of the manganese cluster in vivo. Biochemistry 33: 6137–6149 (1994).Google Scholar
  6. 6.
    Debus RJ: The manganese and calcium ions of photosynthetic oxygen evolution. Biochim Biophys Acta 1102: 269–352 (1992).Google Scholar
  7. 7.
    Deisenhofer J, Epp O, Miki K, Huber R, Michel H: Structure of the protein subunits in the photosynthetic reaction center of Rhodopseudomonas viridis at 3Å resolution. Nature 318: 618–624 (1985).Google Scholar
  8. 8.
    Eggers B, Vermaas WFJ: Truncation of the D2 protein in Synechocystis sp. PCC 6803: a role of the C-terminal domain of D2 in photosystem II function and stability. Biochemistry 32: 11419–11427 (1993).Google Scholar
  9. 9.
    Frankel LK, Bricker TM: Interaction of CPa-1 with the manganese-stabilizing protein of Photosystem II: Identification of domains on CPa-1 which are shielded from N-hydroxysuccinimide biotinylation by the manganese-stabilizing protein. Biochemistry 31: 11059–11064 (1992).Google Scholar
  10. 10.
    Gleiter HM, Haag E, Shen J-R, Eaton-Rye JJ, Inoue Y, Vermaas WFJ, Renger G: Functional characterization of mutant strains of the cyanobacterium Synechocystis sp. PCC 6803 lacking short domains within the large, lumen-exposed loop of the chlorophyll protein CP47 in Photosystem II. Biochemistry 33: 12063–12071 (1994).Google Scholar
  11. 11.
    Haag E, Eaton-Rye JJ, Renger G, Vermaas WFJ: Functionally important domains of the large hydrophilic loop of CP47 as probed by oligonucleotide-directed mutagenesis in Synechocystis sp. PCC 6803. Biochemistry 32: 4444–4454 (1993).Google Scholar
  12. 12.
    Hayatsu H: Bisulfite modification of nucleic acids and their constituents. Prog Nucl Acid Res Mol Biol 16: 75–124 (1976).Google Scholar
  13. 13.
    Kalderon D, Oostra BA, Ely BK, Smith AE: Deletion loop mutagenesis: a novel method for the construction of point mutations using deletion mutants. Nucl Acids Res 10: 5161–5171 (1982).Google Scholar
  14. 14.
    Kuhn MG, Vermaas WFJ: Deletion mutations in a long hydrophilic loop in the photosystem II chlorophyll-binding protein CP43 in the cyanobacterium Synechocystis sp. PCC 6803. Plant Mol Biol 23: 123–133 (1993).Google Scholar
  15. 15.
    Metz JG, Nixon PJ, Rögner M, Brudvig GW, Diner BA: Directed alteration of the D1 polypeptide of PHotosystem II: evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680. Biochemistry 28: 6960–6969 (1989).Google Scholar
  16. 16.
    Miyao M, Murata N: Role of the 33 kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by chloride ions. FEBS Lett 170: 350–354 (1984).Google Scholar
  17. 17.
    Nixon PJ, Diner BA: Analysis of water-oxidation mutants constructed in the cyanobacterium Synechocystis sp. PCC 6803. Biochem Soc Trans 22: 338–343 (1994).Google Scholar
  18. 18.
    Nixon PJ, Diner BA: Aspartate 170 of the Photosystem II reaction center polypeptide D1 is involved in the assembly of the oxygen-evolving manganese cluster. Biochemistry 31: 942–948 (1992).Google Scholar
  19. 19.
    Odom WR, Bricker TM: Interaction of CPa-1 with the manganese-stabilizing protein of Photosystem II: identification of domains cross-linked by 1-ethyl-3-(3-(dimethyl-amino)propyl)carbodiimid. Biochemistry 31: 5616–5620 (1992).Google Scholar
  20. 20.
    Pakrasi HB, Vermaas WFJ: Protein engineering of photosystem II. In: Barber J (ed) The Photosystems: Structure, Function and Molecular Biology, pp. 231–256. Elsevier, Amsterdam (1992).Google Scholar
  21. 21.
    Peden KW, Nathans D: Local mutagenesis within deletion loops of DNA heteroduplexes. Proc Natl Acad Sci USA 79: 7214–7217 (1982).Google Scholar
  22. 22.
    Philbrick JB, Diner BA, Zilinskas BA: Construction and characterization of cyanobacterial mutants lacking the manganese-stabilizing polypeptide of Photosystem II. J Biol Chem 266: 13370–13376 (1991).Google Scholar
  23. 23.
    Pine R, Huang PC: An improved method to obtain a large number of mutants in a defined region of DNA. In: Wu R, Grossman L (eds) Methods in Enzymology vol. 154, pp. 415–430. Academic Press, San Diego, CA (1987).Google Scholar
  24. 24.
    Rögner M, Chisholm DA, Diner BA: Site-directed mutagenesis of the psbC gene of photosystem II: isolation and functional characterization of CP43-less photosystem II core complexes. Biochemistry 30: 5387–5395 (1991).Google Scholar
  25. 25.
    Rutherford AW, Zimmermann J-L, Boussac A: Oxygen evolution. In: Barber J (ed) The Photosystems: Structure, Function and Molecular Biology, pp. 179–229. Elsevier, Amsterdam (1992).Google Scholar
  26. 26.
    Shapiro R, Braverman B, Louis JB, Servis RE: Nucleic acid reactivity and conformation. II. Reaction of cytosine and uracil with sodium bisulfite. J Biol Chem 248: 4060–4064 (1973).Google Scholar
  27. 27.
    Shen G, Eaton-Rye JJ, Vermaas WFJ: Mutation of histidine residues in CP47 leads to destabilization of the photosystem II complex and to impairment of light energy transfer. Biochemistry 32: 5109–5115 (1993).Google Scholar
  28. 28.
    Svensson B, Vass I, Styring S: Sequence of the D1 and D2 reaction center proteins of Photosystem II. Z Naturforsch 46c: 765–776 (1991).Google Scholar
  29. 29.
    Tang XS, Nixon PJ, Britt RD, Diner BA: Coordination and assembly of the manganese cluster, responsible for photosynthetic water oxidation. Photochem Photobiol 59: 82S-83S (1994).Google Scholar
  30. 30.
    van der Bolt F, Vermaas WFJ: Photoinactivation of photosystem II as studied with site-directed D2 mutants of the cyanobacterium Synechosystis sp. PCC 6803. Biochim Biophys Acta 1098: 247–254 (1992).Google Scholar
  31. 31.
    Vermaas WFJ, Styring S, Schröder WP, Andersson B: Photosynthetic water oxidation: the protein framework. Photosynth Res 38: 249–263 (1993).Google Scholar
  32. 32.
    Vermaas WFJ, Charité J, Eggers B: System for site-directed mutagenesis in the psbDI/C operon of Synechocystis sp. PCC 6803. In: Baltscheffsky M (ed) Current Research in Photosynthesis, vol. I, pp. 231–238. Kluwer Academic Publishers, Dordrecht (1990).Google Scholar
  33. 33.
    Vermaas WFJ, Charité J, Shen G: Glu-69 of the D2 protein in photosystem II is a potential ligand to Mn involved in photosynthetic oxygen evolution. Biochemitry 29: 5325–5332 (1990).Google Scholar
  34. 34.
    Vermaas WFJ, Williams JGK, Arntzen CJ: Sequencing and modification of psbB, the gene encoding the CP-47 protein of photosystem II, in the cyanobacterium Synechocystis 6803. Plant Mol Biol 8: 317–326 (1987).Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Svetlana Ermakova-Gerdes
    • 1
    • 2
  • Sergey Shestakov
    • 3
  • Wim Vermaas
    • 1
  1. 1.Department of Botany and Center for the Study of Early Events in PhotosynthesisArizona State UniversityTempeUSA
  2. 2.N. Vavilov Institute of General GeneticsMoscowRussia
  3. 3.N. Vavilov Institute of General GeneticsMoscowRussia

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