Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers

Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug resistance markers, and thus multiple replacements have been restricted. Here, a versatile gene replacement system without this restriction is reported in a unicellular cyanobacterium, Synechococcus sp. PCC 7942. The system is based upon the dominance of a streptomycin-sensitive rps12 gene encoding a ribosomal S12 protein over a streptomycin-resistant rps12-R43 allele with a Lys-43 -->, Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type rps12 gene and a kan gene conferring kanamycin resistance was integrated into the rps12-R43 mutant at the psbAI locus encoding photosystem II D1 protein, resulting in streptomycin-sensitive merodiploids. Despite spontaneous gene conversion in these merodiploids to produce streptomycin-resistant progeny at frequencies ranging from I x 10(-5) to 5 x 10(-5), homologous recombination could be induced by transforming the merodiploids with template plasmids carrying psbAI 5 ' and 3 ' non-coding sequences flanking the D1 coding sequence, which was then replaced by either the gfp ORF for a green fluorescent protein or a precise deletion. Depending on the replication ability of the template plasmids, at most 3-16% of streptomycin-resistant progeny of the merodiploids after transformation were homogenote recombinants with concomitant loss of the kan gene, even in these pollyploid cyanobacteria. The rps12-mediated gene replacement thus makes it possible to construct mutants free from drug resistance markers and opens a way to create cyanobacterial strains bearing an unlimited number of gene replacements.