1887

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, sp. PCC 7942. The system is based upon the dominance of a streptomycin-sensitive gene encoding a ribosomal S12 protein over a streptomycin-resistant allele with a Lys-43→→→Arg substitution. To demonstrate the utility of this method, a cassette consisting of the wild-type gene and a gene conferring kanamycin resistance was integrated into the mutant at the 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 1×10 to 5×10, homologous recombination could be induced by transforming the merodiploids with template plasmids carrying 5′ and 3′ non-coding sequences flanking the D1 coding sequence, which was then replaced by either the 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 gene, even in these polyploid cyanobacteria. The -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.

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2001-08-01
2020-03-29
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