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Plant and Cell Physiology, 2004, Vol. 45, No. 3 333-339
© 2004 Oxford University Press

High-Frequency Gene Replacement in Cyanobacteria Using a Heterologous rps12 Gene

Kazutaka Takahama, Masayoshi Matsuoka1, Kazuhiro Nagahama and Takahira Ogawa

Department of Applied Microbial Technology, Faculty of Engineering, Sojo University, Ikeda 4-22-1, Kumamoto, 860-0082 Japan

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.

1 Corresponding author: E-mail, matsuoka{at}bio.sojo-u.ac.jp; Fax, +81-96-323-1330.


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