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

摘要: Chromosomal gene replacement in cyanobacteria often relies upon the availability of drug resistance markers, and thus multiple replacements have been restricted. Here, a versatile system without this restriction is reported unicellular cyanobacterium, Synechococcus sp. PCC 7942. The based dominance streptomycin-sensitive rps12 encoding ribosomal S12 protein over streptomycin-resistant rps12-R43 allele with Lys-43→→→Arg substitution. To demonstrate utility method, cassette consisting wild-type kan conferring kanamycin was integrated into mutant at psbAI locus photosystem II D1 protein, resulting merodiploids. Despite spontaneous conversion these merodiploids to produce progeny frequencies ranging from 1×10−5 5×10−5, homologous recombination could be induced by transforming template plasmids carrying 5′ 3′ non-coding sequences flanking coding sequence, which then replaced either gfp ORF for green fluorescent or precise deletion. Depending on replication ability plasmids, most 3–16% after transformation were homogenote recombinants concomitant loss gene, even polyploid cyanobacteria. rps12-mediated makes it possible construct mutants free markers opens way create cyanobacterial strains bearing an unlimited number replacements.