Molecular and General Genetics MGG

, Volume 226, Issue 3, pp 401–408 | Cite as

Identification of a genomic region that complements a temperature-sensitive, high CO2-requiring mutant of the cyanobacterium, Synechococcus sp. PCC7942

  • Eiji Suzuki
  • Hideya Fukuzawa
  • Shigetoh Miyachi


In a temperature-sensitive, high CO2-requiring mutant of Synechococcus sp. PCC7942, the ability to fix intracellularly accumulated inorganic carbon was severely impaired at non-permissive temperature (41° C). In contrast, inorganic carbon uptake and ribulose-1,5-bisphosphate carboxylase activity in the mutant were comparable to the respective values obtained with the wild-type strain. The mutant was transformed to the wild-type phenotype (ability to form colonies at non-permissive temperature under ordinary air) with the genomic DNA of the wild-type strain. A clone containing a 36 kb genomic DNA fragment of the wild-type strain complemented the mutant phenotype. The complementing activity region was associated with internal 17 kb SmaI, 15 kb HindIII, 3.8 kb BamHI and 0.87 kb Pstl fragments. These 4 fragments overlapped only in a 0.4 kb HindIII-PstI region. In the transformants obtained with total genomic DNA or a plasmid containing the 3.8 kb BamHI fragment, the ability to fix intracellular inorganic carbon was restored. Southern hybridization and partial nucleotide sequence analysis indicated that the cloned genomic region was located approximately 20 kb downstream from the structural genes for subunits of ribulose-1,5-bisphosphate carboxylase/oxygenase. The cloned region was transcribed into a 0.5 kb mRNA. These results indicate that the cloned genomic region of Synechococcus sp. PCC7942 is involved in the efficient utilization of intracellular inorganic carbon for photosynthesis.

Key words

Complementation analysis Cyanobacteria DNA transformation Inorganic carbon transport hotosynthetic CO2 fixation 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Abe T, Tsuzuki M, Miyachi S (1987) Transport and fixation of inorganic carbon during photosynthesis in cells of Anabaena grown under ordinary air. III. Some characteristics of the HCO3 -transport system in cells grown under ordinary air. Plant Cell Physiol 28:867–874Google Scholar
  2. Abe T, Tsuzuki M, Kadokami Y, Miyachi S (1988) Isolation and characterization of temperature-sensitive, high-CO2 requiring mutant of Anacystis nidulans R2. Plant Cell Physiol 29:1353–1360Google Scholar
  3. Aiba H, Adhya S, De Crombrugghe B (1981) Evidence for two functional gal promoters in intact Escherichia coli cells. J Biol Chem 256:11905–11910Google Scholar
  4. Aizawa K, Miyachi S (1986) Carbonic anhydrase and CO2 concentrating mechanisms in microalgae and cyanobacteria. FEMS Microbiol Rev 39:215–233Google Scholar
  5. Badger MR, Andrews TJ (1982) Photosynthesis and inorganic carbon usage by the marine cyanobacterium, Synechococcus sp. Plant Physiol 70:517–523Google Scholar
  6. Badger MR, Kaplan A, Berry JA (1980) Internal inorganic carbon pool of Chlamydomonas reinhardtii. Evidence for a carbon dioxide-concentrating mechanism. Plant Physiol 66:407–413Google Scholar
  7. Cooper TG, Filmer D (1969) The active species of “CO2” utilized by ribulose diphosphate carboxylase. J Biol Chem 244:1081–1083Google Scholar
  8. Davis GD, Dibner MD, Battey JF (1986) Basic methods in molecular biology. Elsevier, New YorkGoogle Scholar
  9. Friedberg D, Kaplan A, Ariel R, Kessel M, Sejffers J (1989) The 5′-flanking region of the gene encoding the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is crucial for growth of the cyanobacterium Synechococcus sp. strain PCC7942 at the level of CO2 in air. J Bacteriol 171:6069–6076Google Scholar
  10. Golden SS, Brusslan J, Haselkorn R (1987) Genetic engineering of the cyanoacterial chromosome. Methods Enzymol 153:215–231Google Scholar
  11. Hanahan D (1985) Techniques for transformation of E. coli. In: Glover DM (ed) DNA cloning: a practical approach, vol 1. IRL Press, Oxford, pp 109–135Google Scholar
  12. Kaplan A, Badger MR, Berry JA (1980) Photosynthesis and the intracellular inorganic carbon pool in the blue-green alga Anabaena variabilis: response to external CO2 concentration. Planta 149:219–226Google Scholar
  13. Kuhlemeier CJ, Van Arkel GA (1987) Host-vector systems for gene cloning in cyanobacteria. Methods Enzymol 153:199–215Google Scholar
  14. Lau RH, Straus NA (1985) Versatile shuttle cloning vectors for the unicellular cyanobacterium Anacystis nidulans R2. FEMS Microbiol Lett 27:253–256Google Scholar
  15. Mackinney G (1941) Absorption of light by chlorophyll solutions. J Biol Chem 140:315–322Google Scholar
  16. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New YorkGoogle Scholar
  17. Marcus Y, Schwarz R, Friedberg D, Kaplan A (1986) High CO2 requiring mutant of Anacystis nidulans R2. Plant Physiol 82:610–612Google Scholar
  18. Miller AG, Colman B (1980) Active transport and accumulation of bicarbonate by a unicellular cyanobacterium. J Bacteriol 143:1253–1259Google Scholar
  19. Ogawa T, Kaneda T, Omata T (1987) A mutant of Synechococcus PCC7942 incapable of adapting to low CO2 concentration. Plant Physiol 84:711–715Google Scholar
  20. Ogawa T, Williams JGK, Omata T (1990) Molecular analysis of mutants of Synechocystis PCC6803 defective in inorganic carbon transport. In: Baltscheffsky M (ed) Current research in photosynthesis, vol IV. Kluwer Academic Publishers, Dordrecht, pp 471–474Google Scholar
  21. Pierce J, Carlson TJ, Williams JGK (1989) A cyanobacterial mutant requiring the expression of ribulose bisphosphate carboxylase from a photosynthetic anaerobe. Proc Natl Acad Sci USA 86:5753–5757Google Scholar
  22. Price GD, Badger MR (1989a) Ethoxyzolamide inhibition of CO2 uptake in the cyanobacterium Synechococcus PCC7942 without apparent inhibition of internal carbonic anhydrase activity. Plant Physiol 89:37–43Google Scholar
  23. Price GD, Badger MR (1989b) Isolation and characterization of high CO2-requiring mutants of the cyanobacterium Synechococcus PCC7942. Plant Physiol 91:514–525Google Scholar
  24. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467PubMedGoogle Scholar
  25. Schwarz R, Friedberg D, Kaplan A (1988) Is there a role for the 42 kilodalton polypeptide in inorganic carbon uptake by cyanobacteria? Plant Physiol 88:284–288Google Scholar
  26. Shinozaki K, Sugiura M (1983) The gene for the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase is located close to the gene for the large subunit in the cyanobacterium Anacystis nidulans 6301. Nucleic Acids Res 11:6957–5964PubMedGoogle Scholar
  27. Shinozaki K, Yamada C, Takahata N, Sugiura M (1983) Molecular cloning and sequence analysis of the cyanobacterial gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase. Proc Natl Acad Sci USA 80:4050–4054Google Scholar
  28. Shiraiwa Y, Miyachi S (1985) Role of carbonic anhydrase in photosynthesis of blue-green alga (cyanobacterium) Anabaena variabilis ATCC29413. Plant Cell Physiol 26:109–116Google Scholar
  29. Suzuki E, Tsuzuki M, Miyachi S (1987) Photosynthetic characteristics of chloroplasts isolated from Euglena gracillis Z grown photoautotrophically. Plant Cell Physiol 28:1377–1388Google Scholar
  30. Suzuki E, Fukuzawa H, Abe T, Miyachi S (1990) Identification of the genomic region which complements a temperature-sensitive, high-CO2 requiring mutant of the cyanobacterium, Synechococcus PCC7942. In: Baltscheffsky M (ed) Current research in photosynthesis, vol IV. Kluwer Academic Publishers, Dordrecht, pp 467–470Google Scholar
  31. Volokita M, Zenvirth D, Kaplan A, Reinhold L (1984) Nature of the inorganic carbon species actively taken up by the cyanobacterium Anabaena variabilis. Plant Physiol 76:599–602Google Scholar
  32. Williams JGK, Szalay AA (1983) Stable integration of foreign DNA into the chromosome of the cyanobacterium Synechococcus R2. Gene 24:37–51Google Scholar
  33. Yanisch-Perron C, Vieira J, Messing J (1985) Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103–119CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 1991

Authors and Affiliations

  • Eiji Suzuki
    • 1
  • Hideya Fukuzawa
    • 1
  • Shigetoh Miyachi
    • 1
  1. 1.Institute of Applied MicrobiologyUniversity of TokyoBunkyo-ku, TokyoJapan

Personalised recommendations