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Transformation and gene replacement in the facultatively chemoheterotrophic, unicellular cyanobacterium Synechocystis sp. PCC6714 by electroporation

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Abstract

The unicellular cyanobacterium Synechocystis sp. PCC6714 can grow not only under photoautotrophic conditions, but also under chemoheterotrophic conditions if glucose is added to the medium. This makes it useful for the study of many aspects of bioenergetic mechanisms. In contrast to its closely related strain Synechocystis sp. PCC6803, which cannot grow chemoheterotrophically, Synechocystis PCC6714 is not naturally transformable. To enable gene transfer in this strain, we established a method for the introduction of self-replicating IncQ plasmids and for gene replacement using electroporation.

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Acknowledgments

We thank Dr. Jörg Burgstaller, Dr. Elisabeth Hofmann, and Ms. Elisabeth Riegel for their assistance in PCR, DNA cloning, and sequencing.

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Authors and Affiliations

  1. Institute of Animal Breeding and Genetics, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, Austria

    Alfred Ludwig & Thomas Czerny

  2. Molecular & Cell Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Rd., La Jolla, CA, 92037, USA

    Thomas Heimbucher

  3. Research Group of Molecular Pharmacology and Toxicology, Department of Science, University of Veterinary Medicine, Veterinärplatz 1, Vienna, 1210, Austria

    Wolfgang Gregor

  4. Institute of Physical Chemistry, University of Vienna, Alhanstraße 14, Vienna, 1090, Austria

    Georg Schmetterer

Authors
  1. Alfred Ludwig
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  2. Thomas Heimbucher
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  3. Wolfgang Gregor
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  4. Thomas Czerny
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  5. Georg Schmetterer
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Corresponding author

Correspondence to Alfred Ludwig.

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Fig. S1a

Plasmid constructions. pKT210 (Haring et al. 1985) was cut with PstI, the fragments were separated, and the plasmid lacking the chloramphenicol resistance cassette was religated to obtain pALUV6. The psbA1 sequence of Synechocystis sp. PCC6714 was amplified by PCR with primers containing an EcoRI and a BamHI site, respectively. The obtained PCR product and pUC19 were both cut with EcoRI and BamHI, and the psbA1-fragment was ligated into pUC19 yielding pALUV18. pALUV18 was cut with BsrGI and NsiI, and the nptII gene (originally from Tn903) was cut from pRL446 with KpnI and PstI, two enzymes that are compatible with BsrGI and NsiI. The nptII fragment was ligated into pALUV18 to obtain pALUV18K. (TIFF 108 kb)

Fig. S1b

Plasmid construction. The cytM sequence of Synechocystis sp. PCC6714 was amplified by PCR using primers containing an EcoRI and a BamHI restriction site, respectively. The PCR product and pUC19 were both cut with these two enzymes, and the cytM fragment was ligated into pUC19 to obtain pALUV17. pALUV17 was cut with BsaAI, and the cat-gene (originally from Tn9) was cut out from pJM8 with BamHI. 5′-Overhangs were refilled with T4 polymerase, and the cat fragment was ligated into pALUV17 yielding pALUV17CR. (TIFF 290 kb)

Fig. S2a

Sequence alignment. “our sequence” corresponds to the psbA1 fragment from the Synechocystis sp. PCC6714 strain with the cytM gene disrupted by the cat gene introduced by electroporation with pALUV17CR. emb∣X15514.1 is the accession number of the psbA1 gene from Synechocystis sp. PCC6714 shown here. (TIFF 205 kb)

Fig. S2b

Sequence alignment. “our mutant” corresponds to the disrupted cytM gene by electroporation of Synechocystis sp. PCC6714 with pALUV17CR. emb∣X82563.1 is the accession number of the cytM gene from Synechocystis sp. PCC6714 shown here. VOO622 is the accession number of the cat gene from Tn9 shown here. (TIFF 183 kb)

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Ludwig, A., Heimbucher, T., Gregor, W. et al. Transformation and gene replacement in the facultatively chemoheterotrophic, unicellular cyanobacterium Synechocystis sp. PCC6714 by electroporation. Appl Microbiol Biotechnol 78, 729–735 (2008). https://doi.org/10.1007/s00253-008-1356-y

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