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Volume 147, Issue 8

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

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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Keyword(s): Apr, ampicillin-resistant , Cmr, chloramphenicol-resistant , Cms, chloramphenicol-sensitive , gene conversion , homologous recombination , Kmr, kanamycin-resistant , Kms, kanamycin-sensitive , merodiploid , psbAI , Smr, streptomycin-resistant , Strr, streptomycin-resistant (chromosomal) , Strs, streptomycin-sensitive (chromosomal) and Synechococcus
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2001-08-01
2024-04-18
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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
Microbiology 147, 2077 (2001); https://doi.org/10.1099/00221287-147-8-2077
/content/journal/micro/10.1099/00221287-147-8-2077
/content/journal/micro/10.1099/00221287-147-8-2077
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