Targeting mutations to specific genomic loci is invaluable for assessing in vivo the effect of these changes on the biological role of the gene in study. Here, we attempted to introduce a mutation that was previously implicated in an increased heat stability of the mesophilic cyanobacterium Synechocystis sp. PCC6803 via homologous recombination to the psbA gene of Chlamydomonas reinhardtii. For that, we established a strategy for targeted mutagenesis that was derived from the efficient genome-wide homologous-recombination-based methodology that was used to target individual genes of Saccharomyces cerevisiae. While the isolated mutants did not show any benefit under elevated temperature conditions, the new strategy proved to be efficient for C. reinhardtii even in the absence of direct positive selection.

Department of Phototrophic Microorganisms Institute of Microbiology CAS Novohradská 237 37981 Třeboň Czech Republic

Department of Plant and Environmental Sciences Weizmann Institute of Science Herzl 234 7610001 Rehovot Israel

Faculty of Science University of South Bohemia in České Budějovice Branišovská 1645 31 A 37005 České Budějovice Czech Republic

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