title: Gene replacement in cyanobacteria mediated by a dominant streptomycin-sensitive rps12 gene that allows selection of mutants free from drug resistance markers.
abstract: 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 1x10(-5) to 5x10(-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 polyploid 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.
authors.affiliation: Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan1.
authors.forename: Masayoshi
authors.initials: M
authors.lastname: Matsuoka
authors.affiliation: Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan1.
authors.forename: Kazutaka
authors.initials: K
authors.lastname: Takahama
authors.affiliation: Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan1.
authors.forename: Takahira
authors.initials: T
authors.lastname: Ogawa
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000458:Cyanobacteria / Q000187:drug effects* / Q000235:genetics*; D004352:Drug Resistance, Microbial / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial; D005799:Genes, Dominant; D005819:Genetic Markers; D045322:Photosynthetic Reaction Center Complex Proteins / Q000235:genetics; D045332:Photosystem II Protein Complex; D010940:Plant Proteins / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D011995:Recombination, Genetic; D013307:Streptomycin / Q000494:pharmacology*; D014169:Transformation, Bacterial
chemical_list: D000900:Anti-Bacterial Agents; D005819:Genetic Markers; D045322:Photosynthetic Reaction Center Complex Proteins; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; C103919:Rps12 protein, plant; D013307:Streptomycin