High-frequency gene replacement in cyanobacteria using a heterologous rps12 gene.
Multiple targeted gene replacements are often required for functional analyses of cyanobacterial genomes. For this purpose, we previously devised a simple genetic method, termed rps12-mediated gene replacement, in a cyanobacterium Synechococcus elongatus PCC 7942 for construction of mutants free from drug resistance markers. Here, we improved the method by employing a heterologous rps12 gene encoding a ribosomal protein S12 from Synechocystis sp. PCC 6803. Dominant streptomycin-sensitive phenotype of the Synechocystis rps12 gene was manifested only when it was expressed under the strong promoter of psbAI gene in S. elongatus PCC 7942 bearing a streptomycin-resistant rps12 allele. Transformation of the rps12 heteroallelic strains with non-replicating template plasmids permitted the selection of recombinants with gene replacement at frequencies up to 50% among streptomycin-resistant progeny.
Takahama
Kazutaka
K
Department of Applied Microbial Technology, Faculty of Engineering, Sojo University, Ikeda 4-22-1, Kumamoto, 860-0082 Japan.
Matsuoka
Masayoshi
M
Nagahama
Kazuhiro
K
Ogawa
Takahira
T
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001483:Base Sequence; D000458:Cyanobacteria / Q000187:drug effects / Q000235:genetics*; D024881:Drug Resistance, Bacterial / Q000235:genetics; D017353:Gene Deletion; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics*; D011995:Recombination, Genetic; D012269:Ribosomal Proteins / Q000235:genetics*; D013045:Species Specificity; D013307:Streptomycin / Q000494:pharmacology; D014169:Transformation, Bacterial
D000900:Anti-Bacterial Agents; D012269:Ribosomal Proteins; C034212:ribosomal protein S12; D013307:Streptomycin