The cyanobacterium Synechocystis sp. PCC 6803 was the first photosynthetic organism for which a complete genome sequence became available (Kaneko et al., 1996). The organism has a genome size of 3.57 Mbp containing close to 3200 open reading frames. All genomic information regarding this organism is available on a dedicated web site named CyanoBase (http://www.kazusa.or.jp/cyano/cyano.html). A very attractive feature of this cyanobacterium is that it is spontaneously transformable and integrates foreign DNA by homologous double-recombination, thus enabling the facile generation of mutants lacking specific genes. As the Synechocystis sp. PCC 6803 genome sequence has been available for many years, it has become an organism of choice for many groups and consequently a large amount of information has been accumulated on the function of its open reading frames. Web-based information on the function of a number of open reading frames can be found at CyanoGenes (http://www.kazusa.or.jp/cyano/Synechocystis/comments/) and CyanoMutants (http://www.kazusa.or.jp/cyano/Synechocystis/mutants/), which are sub-sites of CyanoBase. Another very useful site is the Synechocystis gene annotation database, CyORF (http://cyano.genome.ad.jp/).

     Testing of a putative function of an ORF usually is best carried out by targeted deletion mutagenesis: homologous double recombination between a plasmid construct and the Synechocystis genome is very efficient. Synechocystis sp. PCC 6803 is transformed with a gene deletion construct containing Synechocystis sequence from up- and downstream of the gene of interest, but with at least part of the gene itself replaced by an antibiotic-resistance cassette: transformation entails addition of DNA to a 0.1-0.5 ml volume of concentrated Synechocystis culture, followed by incubation for 1-6 hours and plating out. After 20-24 hours selective conditions can be applied. Colonies of transformants come up in about one week, and can then be restreaked on plates with increasingly higher concentration of the antibiotic for which a resistance marker has been introduced. Synechocystis sp. PCC 6803 carries multiple genome copies per cell. To obtain a pure mutant phenotype, all wild-type genome copies need to be replaced. Two factors are important to readily obtain segregation: (1) a gradual increase in antibiotic selection pressure, and (2) selection of growth conditions under which the mutant phenotype has a competitive advantage or is not very much impaired in comparison with the wild type. A convenient and rapid method to screen for segregation of wild-type and mutant genotypes is to prepare DNA from propagated transformants and to amplify the region of the mutation by PCR.

     A gene deletion (or mutation) procedure is appropriate when hypotheses regarding gene function are tested, and the Vermaas lab continues to contribute examples of elucidation of Synechocystis gene function (see publications for selected examples). However, if a putative function of an ORF cannot be predicted, gene function discovery is more complicated. One approach is to isolate natural mutants that have gained the potential to grow under restrictive conditions. With the sequencing of the Synechocystis sp. PCC 6803 genome the locus of the mutation can be mapped simply by a complementation experiment (Vermaas, 1998). After isolation of DNA from the mutant, it is cut with any 5-10 of the 16 restriction enzymes for which a restriction map of the Synechocystis sp. PCC 6803 genome has been constructed, and then DNA samples digested with one of these enzymes are size-separated on a gel. The lanes of the gel are cut into 20-25 fractions, each corresponding to a size category between about 1 and 20 kbp, and each fraction is put into an eppendorf tube. The gel pieces are frozen and thawed, and then spun down in a microcentrifuge. The supernatant liquid contains a significant amount of DNA, which can be precipitated or used directly for transformation of the parental strain plated on medium that will support growth of the mutant but not of the parental strain. The size range of the collection of restriction fragments that leads to functional complementation can be determined for each restriction enzyme. We have made a list of genome locations of restriction sites and of the corresponding restriction fragment sizes (ordered by size) for the 16 restriction enzymes. By determining which genome location(s) are present in all restriction fragment collections that complement a unique solution of a 0.5-3 kbp region carrying the mutation locus usually can be found when using only 5 different restriction enzymes. The Kaleidagraph files with the 16 Synechocystis sp. PCC 6803 restriction maps and size-sorted restriction fragment tables are available upon request.


References
 
  • Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosawa, M., Sugiura, M., Sasamoto, S., Kimura, T., Hosochi, T., Matsuno, A., Muraki, A., Nakazaki, N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M., Yasuda, M. and Tabata, S. (1996). Sequence analysis of the genome of the unicellular cyanobacterium Synechocystis sp. strain PCC 6803. II. Sequence determination of the entire genome and assignment of potential protein-coding regions. DNA Research 3, 109-136 and 185-209 (supplement).

  • Vermaas, W.F.J. (1998) Gene modifications and mutation mapping to study the function of photosystem II. Meth. Enzymol. 297, 293-310.

Publications
 
 

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