Small, extrachromosomal DNA from Spirulina platensis: Characterization and application

Spirulina is one of the most commercially important microalgae and promises to provide wider applications if it is genetically manipulated for producing a desired substance. However, there has so far been no host-vector system for stable transformation. One of the authors here reported the presence of cryptic plasmids in Spirulina platensis F3 strain (QIN Song et al., Chin J. Oceanol. Limnol., 11,285 (1993)). We have now cloned two DNA species from a similar strain, determined their whole sequence, and attempted to construct a vector for S. platensis. The intact form of the two DNA species in a cell was linear, which was found from the comparison between the mobility in gel electrophoresis and their sequences. The DNA size of the two species was 1111 bp (designated FS-1) and 356 bp (FS-2), respectively. The DNA sequence data revealed that both contained many direct and inverted repeats. Neither species has an ORF of reasonable size and its leading sequence. The number of copies per cell was estimated to be less than one in both species. Seemingly they have no replication origin. Therefore it is unlikely that they are plasmid. They had little similarity to transposon in their sequence structure. We detected the same sequence in the chromosomal DNA by PCR and by Southern hybridization. Another peculiar phenomenon was that a FS-1 contained plasmid (bluescript II SK(-), Stratagene) reduced its copy number per cell to less than one tenth of that of normal plamid in Escherichia coli (JM109). The reverse orientation of FS-1 in the vector gave the same reduction. At the present we rather think that they may not be a biological entity, but just a DNA fragment released from the chromosomal DNA during replication and survived temporally. We may, however, expect their ability of efficient integration into the chromosomal DNA, based on many direct and inverted repeats. A research is in progress towards to recombination of Spirulina with luxAB, a reporter gene.