Gene targeting in cyanobacteria utilizing I-TevI endonuclease

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Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug-resistant markers, and thus multiple replacements have been restricted. We report here a versatile gene replacement system without this restriction in a unicellular cyanobacterium Synechococcus sp. PCC 7942. The strategy is based upon the dominance of streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with Lys43Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps 12 gene and a kan gene conferring kanamycin resistance was integrated in the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite a spontaneous gene conversion in these merodiploids to form streptomycin-resistant progeny at frequencies ranging from 1 x 10^<-5> to 5 x 10^<-5>, we could induce homologous recombination by transforming the merodiploids with template plasmids carrying psbAI 5' and 3' non-coding sequences flanking the D1-coding sequence which was replaced by either gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3 to 16 % of streptomycin-resistant progeny from the merodiploids after transformation turned out homogenote recombinants with concomitant loss of the kan gene even for polyploid cyanobacteria. The rps 72-mediated gene replacement thus makes it possible to construct mutants free from drug-resistant markers, and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.