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

Department of Applied Microbial Technology, Sojo University, Ikeda 4-22-1, Kumamoto 860-0082, Japan¹

Author for correspondence: Masayoshi Matsuoka. Tel: +81 96 326 3111. Fax: +81 96 323 1330. e-mail: matsuoka{at}bio.sojo-u.ac.jp

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-43Arg 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.

Keywords: homologous recombination, Synechococcus, gene conversion, merodiploid, psbAI

Abbreviations: Ap^r, ampicillin-resistant; Cm^r, chloramphenicol-resistant; Cm^s, chloramphenicol-sensitive; Km^r, kanamycin-resistant; Km^s, kanamycin-sensitive; Sm^r, streptomycin-resistant; Str^r, streptomycin-resistant (chromosomal); Str^s, streptomycin-sensitive (chromosomal)

This article has been cited by other articles:

				K. Takahama, M. Matsuoka, K. Nagahama, and T. Ogawa High-Frequency Gene Replacement in Cyanobacteria Using a Heterologous rps12 Gene Plant Cell Physiol., March 15, 2004; 45(3): 333 - 339. [Abstract] [Full Text] [PDF]