Photosynthesis Research

, Volume 107, Issue 2, pp 215–221 | Cite as

One-step plasmid construction for generation of knock-out mutants in cyanobacteria: studies of glycogen metabolism in Synechococcus sp. PCC 7002

  • Jacob H. Jacobsen
  • Lisa Rosgaard
  • Yumiko Sakuragi
  • Niels-Ulrik Frigaard
Technical Communication


Genome sequences of microorganisms typically contain hundreds of genes with vaguely defined functions. Targeted gene inactivation and phenotypic characterization of the resulting mutant strains is a powerful strategy to investigate the function of these genes. We have adapted the recently reported uracil-specific excision reagent (USER) cloning method for targeted gene inactivation in cyanobacteria and used it to inactivate genes in glycogen metabolism in Synechococcus sp. PCC 7002. Knock-out plasmid constructs were made in a single cloning step, where transformation of E. coli yielded about 90% colonies with the correct construct. The two homologous regions were chosen independently of each other and of restriction sites in the target genome. Mutagenesis of Synechococcus sp. PCC 7002 was tested with four antibiotic resistance selection markers (spectinomycin, erythromycin, kanamycin, and gentamicin), and both single-locus and double-loci mutants were prepared. We found that Synechococcus sp. PCC 7002 contains two glycogen phosphorylases (A0481/glgP and A2139/agpA) and that both need to be genetically inactivated to eliminate glycogen phosphorylase activity in the cells.


Gene inactivation Glycogen metabolism Glycogen phosphorylase Homologous recombination Natural transformation Uracil-specific excision reagent 

















Uracil-specific excision reagent



This work was supported by a grant from the Danish National Advanced Technology Foundation (HTF) to N.-U. F. and a grant from the Danish Council for Independent Research | Natural Sciences (FNU) and Fuel-4-Life Consortium (University of Copenhagen) to Y. S.

Supplementary material

11120_2010_9613_MOESM1_ESM.pdf (2.5 mb)
Supplementary material 1 (PDF 2534 kb)


  1. Atsumi S, Higashide W, Liao JC (2009) Direct photosynthetic recycling of carbon dioxide to isobutyraldehyde. Nat Biotechnol 27(12):1177–1180. doi: 10.1038/nbt.1586 CrossRefPubMedGoogle Scholar
  2. Becker A, Schmidt M, Jager W, Puhler A (1995) New gentamicin-resistance and lacZ promoter-probe cassettes suitable for insertion mutagenesis and generation of transcriptional fusions. Gene 162(1):37–39. doi: 10.1016/0378-1119(95)00313-U CrossRefPubMedGoogle Scholar
  3. Bibel M, Brettl C, Gosslar U, Kriegshauser G, Liebl W (1998) Isolation and analysis of genes for amylolytic enzymes of the hyperthermophilic bacterium Thermotoga maritima. FEMS Microbiol Lett 158(1):9–15. doi: 10.1016/S0378-1097(97)00420-5 CrossRefPubMedGoogle Scholar
  4. Bitinaite J, Rubino M, Varma KH, Schildkraut I, Vaisvila R, Vaiskunaite R (2007) USER friendly DNA engineering and cloning method by uracil excision. Nucleic Acids Res 35(6):1992–2002. doi: 10.1093/nar/gkm041 CrossRefPubMedGoogle Scholar
  5. Clerico EM, Ditty JL, Golden SS (2007) Specialized techniques for site-directed mutagenesis in cyanobacteria. Methods Mol Biol 362:155–171. doi: 10.1007/978-1-59745-257-1_11 CrossRefPubMedGoogle Scholar
  6. Elhai J, Wolk CP (1988) A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers. Gene 68(1):119–138. doi: 10.1016/0378-1119(88)90605-1 CrossRefPubMedGoogle Scholar
  7. Flores E, Muro-Pastor A, Meeks J (2008) Gene transfer to cyanobacteria in the laboratory and in nature. In: Herrero A, Flores E (eds) The cyanobacteria: molecular biology, genomics and evolution. Caister Academic Press, Norfolk, pp 45–57Google Scholar
  8. Frandsen RJ, Andersson JA, Kristensen MB, Giese H (2008) Efficient four fragment cloning for the construction of vectors for targeted gene replacement in filamentous fungi. BMC Mol Biol 9:70. doi: 10.1186/1471-2199-9-70 CrossRefPubMedGoogle Scholar
  9. Frigaard NU, Sakuragi Y, Bryant DA (2004) Gene inactivation in the cyanobacterium Synechococcus sp. PCC 7002 and the green sulfur bacterium Chlorobium tepidum using in vitro-made DNA constructs and natural transformation. Methods Mol Biol 274:325–340. doi: 10.1385/1-59259-799-8:325 PubMedGoogle Scholar
  10. Fu J, Xu X (2006) The functional divergence of two glgP homologues in Synechocystis sp. PCC 6803. FEMS Microbiol Lett 260(2):201–209. doi: 10.1111/j.1574-6968.2006.00312.x CrossRefPubMedGoogle Scholar
  11. Geu-Flores F, Nour-Eldin HH, Nielsen MT, Halkier BA (2007) USER fusion: a rapid and efficient method for simultaneous fusion and cloning of multiple PCR products. Nucleic Acids Res 35(7):e55. doi: 10.1093/nar/gkm106 CrossRefPubMedGoogle Scholar
  12. Gulig PA, Tucker MS, Thiaville PC, Joseph JL, Brown RN (2009) USER friendly cloning coupled with chitin-based natural transformation enables rapid mutagenesis of Vibrio vulnificus. Appl Environ Microbiol 75(15):4936–4949. doi: 10.1128/AEM.02564-08 CrossRefPubMedGoogle Scholar
  13. Herrero A, Flores E (2008) The cyanobacteria: molecular biology, genomics, and evolution. Caister Academic Press, NorfolkGoogle Scholar
  14. Holkenbrink C, Ocón Barbas S, Mellerup A, Otaki H, Frigaard N-U (2011) Sulfur globule oxidation in green sulfur bacteria is dependent on the dissimilatory sulfite reductase system. Microbiology (in press)Google Scholar
  15. Lee JM, Ryu JY, Kim HH, Choi SB, de Marsac NT, Park YI (2005) Identification of a glucokinase that generates a major glucose phosphorylation activity in the cyanobacterium Synechocystis sp. PCC 6803. Mol Cells 19(2):256–261PubMedGoogle Scholar
  16. Nour-Eldin HH, Hansen BG, Norholm MH, Jensen JK, Halkier BA (2006) Advancing uracil-excision based cloning towards an ideal technique for cloning pcr fragments. Nucleic Acids Res 34(18):e122. doi: 10.1093/nar/gkl635 CrossRefPubMedGoogle Scholar
  17. Nour-Eldin HH, Geu-Flores F, Halkier BA (2010) User cloning and user fusion: the ideal cloning techniques for small and big laboratories. Methods Mol Biol 643:185–200. doi: 10.1007/978-1-60761-723-5_13 CrossRefPubMedGoogle Scholar
  18. Richmond A (2004) Handbook of microalgal culture: biotechnology and applied phycology. Blackwell Science, Oxford/AmesGoogle Scholar
  19. Sakuragi Y, Maeda H, Dellapenna D, Bryant DA (2006) Alpha-tocopherol plays a role in photosynthesis, macronutrient homeostasis of the cyanobacterium Synechocystis sp. PCC 6803 that is independent of its antioxidant function. Plant Physiol 141(2):508–521. doi: /10.1104/pp.105.074765 CrossRefPubMedGoogle Scholar
  20. Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory, Cold Spring HarborGoogle Scholar
  21. Stevens S Jr, Patterson C, Myers J (1973) The production of hydrogen peroxide by blue-green algae: a survey. J Phycol 9(4):427–430. doi: 10.1111/j.1529-8817.1973.tb04116.x Google Scholar
  22. Tripp HJ, Bench SR, Turk KA, Foster RA, Desany BA, Niazi F, Affourtit JP, Zehr JP (2010) Metabolic streamlining in an open-ocean nitrogen-fixing cyanobacterium. Nature 464:90–94. doi: 10.1038/nature08786 CrossRefPubMedGoogle Scholar
  23. Wang HL, Postier BL, Burnap RL (2002) Optimization of fusion PCR for in vitro construction of gene knockout fragments. Biotechniques 33(1):26–32PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Jacob H. Jacobsen
    • 1
  • Lisa Rosgaard
    • 2
  • Yumiko Sakuragi
    • 2
  • Niels-Ulrik Frigaard
    • 1
  1. 1.Department of BiologyUniversity of CopenhagenCopenhagenDenmark
  2. 2.Laboratory for Molecular Plant Biology, VKR Research Centre Pro-Active Plants, Department of Plant Biology and BiotechnologyUniversity of CopenhagenFrederiksbergDenmark

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