Journal Article

High-Frequency Gene Replacement in Cyanobacteria Using a Heterologous <i>rps12</i> Gene

Kazutaka Takahama, Masayoshi Matsuoka, Kazuhiro Nagahama and Takahira Ogawa

in Plant and Cell Physiology

Published on behalf of Japanese Society of Plant Physiologists

Volume 45, issue 3, pages 333-339
Published in print March 2004 | ISSN: 0032-0781
Published online March 2004 | e-ISSN: 1471-9053 | DOI: http://dx.doi.org/10.1093/pcp/pch041
High-Frequency Gene Replacement in Cyanobacteria Using a Heterologous rps12 Gene

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  • Biochemistry
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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.

Keywords: Keywords: Mutation — psbAI promoter — Ribosomal protein — Streptomycin — Synechococcus elongates.; Abbreviations: Ampr, ampicillin resistance; gfp, green fluorescent protein; kan, kanamycin resistance; Kmr, kanamycin-resistant; Kms, kanamycin-sensitive; ORF, open reading frame; Strr, streptomycin-resistant; Strs, streptomycin-sensitive.

Journal Article.  3949 words.  Illustrated.

Subjects: Biochemistry ; Molecular and Cell Biology ; Plant Sciences and Forestry

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